Lasers in Dermatology PDF

Foreword by the late
Dr. Leon Goldman
LASERS
IN
MEDICINE
Edited by
Ronald W. Waynant
CRC PR E S S
Boca Raton London New York Washington, D.C.
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Library of Congress Cataloging-in-Publication Data
Lasers in Medicine / edited by Ronald W. Waynant.

p. ; cm.
Includes bibliographical references and index.
ISBN 0-8493-1146-2 (alk. paper)
1. Lasers in medicine. 2. Lasers in surgery. I. Waynant, Ronald W.
[DNLM: 1. Lasers. 2. Keratomileusis, Laser In Situ. 3. Laser Coagulation. 4. Laser
Surgery. 5. Microscopy, Confocal. WB 117 L34341 2001[
R857.L37L382 2001
610’.28--dc21 2001043530
CIP
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Dedication
To my mother, father, wife and children

for their patience with this project. Never has a book
required so much.
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Foreword
The Early Development

It is curious that the early interest in this darling of the physicists called a laser focused on concerns
about safety. With memories of the hazards of x-ray development, it was not known what this powerful
light could do. In fact, the early worries regarding laser applications in ophthalmology were that there
would be x-ray changes in the eyes that could be dangerous. Focusing on the hazards to the eye, our
interest was with the dangers to the workmen who were building lasers. With that idea, we established
the first comprehensive laser laboratory for medicine at the University of Cincinnati in 1961. The
development of a safety program for the use of lasers in medicine was the first goal.
It was fortunate that the laser in the visible light range, the ruby, was the first laser we used. The ruby
laser furnished the operating surgeon with a special scalpel that was related to the color of the target
area. The interest in these studies was tremendous, so funding was given by the John A. Hartford
Foundation for the development of the extensive laser research laboratory at the Children’s Hospital of
the Medical Center of the University of Cincinnati. This permitted a number of disciplines in medicine
and biology to do basic work, first with the ruby laser, followed by the CO2 laser and the argon laser,
and, in 1962, the development of the first Q-switched ruby laser.
After several years, the Laser Research Laboratory at the Children’s Hospital was moved to the Depart-
ment of Dermatology, College of Medicine, University of Cincinnati. This laboratory continued to be
used for basic biomedical and then clinical applications in many areas of surgery. The Laser Research
Laboratory also developed practical laser surgery teaching courses for hands-on learning with an emphasis
on controls. These courses continue today with more or fewer controls, relatively few with “hands-on
learning” — some even of value for the expenses required. In 1993, Puliafito introduced a teaching course
with diagnostics and design engineering in addition to laser surgery.
Throughout the years, the programs of research and development continued and new applications
were discovered, which led to the development of laser biomedical institutes and the laser biomedical
industry. The laser biomedical industry even today is called a “profitless profit organization.” This is
because laser biomedical instrumentation is considered a form of technology development rather than
of market development. The “hazards” that the laser biomedical industry might be influenced by vivid
market developments have been shown recently in two areas: one, the development of laparoscopic
cholecystectomy and, second, the renaissance of laser dentistry, which we first established in the 1960s.
For laparoscopic cholecystectomy, the marketing influence initially recommended the use of expensive
laser systems, discouraging the general surgeons, who had been the last group of conservative medical
disciplines attracted to the laser. As a result, untrained surgeons ventured into this high surgical tech-
nology for laparoscopic cholecystectomy — at the time, a new ambulatory technology that had been
treated conservatively over a period of years. The application of this new surgical technology proceeded
without the continued development of controls, basic training and safety courses, which, in turn, led to
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disappointments, concerns, and reactions. However, the result was an over-enthusiasm for the short
morbidity associated with gallbladder operations. The belated application of control electrosurgery led
to some disenchantment with laparoscopic laser surgery among general surgeons and even the public.
However, a small sampling of surgeons continued to use laparoscopic laser surgery and, eventually, its
use expanded to the appendectomy, hiatus hernia and herniorrhaphy. Again, the need for controls and
even the actual need for such surgery were the driving forces behind the developing technology.
A similar “vivid” market episode led to the revival of laser dentistry, which had been neglected because
of disinterest among dentists, the expense of instrumentation for general practice and the lack of con-
tinued research in that field after the 1960s. The result of this market influence was the increased sales
of Nd:YAG lasers; expensive and not necessary for common soft tissue dental surgery. This led to the
purchase of unwanted expensive instruments that the practicing dentist could not afford. The current
revival of basic research and continued interest in CO2 and holmium lasers for operative dentistry are
gradually rebuilding the confidence of dental surgeons in particular, and the continued research on
inexpensive laser diagnostics is influencing even regular dental practitioners.
The Current Laser Theme

The rapid production of laser biomedical instrumentation, the recognition of the importance of laser
research in the laboratory and beyond, as well as the influence of laser biomedical engineers are all
characteristics of the modern scene. Efforts to develop the new comprehensive laser engineering programs
in a formal style have not been possible. So-called optical engineering is provided mostly in short courses
in postgraduate engineering programs — few “real” programs in optical engineering are available. The
original proposal of laser engineering included, first, two years of basic optical-, electrical- and mechan-
ical-engineering courses, then cooperative laser engineering for the subsequent three years.
Cooperative engineering, first established by Herman Schneider, Dean of the College of Engineering
at the University of Cincinnati, means one semester at the College of Engineering alternating with the
next semester in industry. For laser engineering, this “industry” meant factory laser production, laser
applications in the military or the laser biomedical industry. An alternate worker held the student’s job
position while he or she returned to college. Cooperative engineering also permitted more students to
attend engineering college through their jobs in industry. Briefly, the contact with the actual laser industry
during the engineering training period made for a practical insight into what needed to appear on the
floor of the factory.
The problem here, of course, is finding faculty for the third, fourth and fifth years at the College of
Engineering. It is the obligation of the laser industry, the military and the biomedical groups to help
supply such faculties at both undergraduate and graduate levels. Leroy Hood, M.D., Gates Professor and
Chairman of the Department of Biotechnology at the University of Washington School of Medicine,
believes that, “The future problems of biology will primarily have to do with the analyses of complex
systems and networks and, accordingly, we must develop tools to handle these complex systems. The
development of these tools will require the joining of applied science, engineering and other disciplines
to biology to develop the complex tools of the future. We see a special role in developing new compu-
tational tools in both hardware and software areas. My feeling is that lasers would play a very important
role in the development of new biological and medical technologies.”
There is a need for the use of the consortium in laser biomedical technology; the need for cooperative
groups to work together to solve difficult problems is important. As indicated, this has not been possible
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in the United States, whereas it is possible, with government help, in Japan. The only consortium presently
available in the United States now seems to be the automobile consortium for the electric automobile.
The secrecy and competition of the laser biomedical industry has made it difficult to develop a consortium.
The Future
Our usual biomedical lasers are slowly becoming smaller, more flexible, and somewhat less expensive.
Technology is producing new lasers with more Q-switched systems, which brings about special safety
problems such as more viable plume fragments. In general, the new lasers are being developed for
specific applications.
Our interest in the future of laser biomedical instrumentation is to consider development of multi-
wave systems. This is to attempt to have more applications in single or compact unit systems. We survey
the initial development of the following multi-wave systems:
• R. Rox Anderson suggests the economical junction diode systems alone or pumping solid state lasers.
• Multiple heavy metal vapor systems, which we have just started.
• Multi-wavelength systems of the parametric oscillator types such as the MOPO of Spectra Physics
from the third harmonic of Nd:YAG with 200 nm up to 4500 nm; Continuum with the second
harmonic and a smaller number of wavelengths. Coherent Laser Co. is also considering a para-
metric oscillator system; the type is not known.
Our program at the Naval Medical Center is to develop a seven-wavelength system for our needs,
such as for blood vessel disorders, for pigmentations, for tattoo removal, for cancer, and for localized
refractory dermatologic conditions. For that, we are working through J.W. Steger, CAPT MC USN,
with the copper vapor laser pumping a ruby laser without Q-switching, 513, 578, 513 + 578, and 694
in the nanosecond range. This combination gives us four wavelengths. Then, with another heavy metal
vapor laser, barium, we are able to have 1500, 1300, and a third wavelength of a mixture of the 1500
and 1300. Also, the copper vapor laser can pump gold and pump lead, giving us additional wavelengths.
A third area of multiple laser systems is the MOPO 700 series, as indicated, of Spectra Physics, which
can provide wavelengths from the 200 nm to the 4500 nm range with millijoule output. With the
biomedical engineering developments for instrumentation of this latter group, we see the future of
laser biomedical instrumentation. Research and development is just starting. The complex problem
of laser safety for such a system is now with development by Rockwell. It is difficult in this current
era, at least in the United States, with concerns for the effects of new programs on health costs, for
continued basic and applied laser research.
Conclusions
There is proof that laser surgery reduces the cost of in-patient surgery, a significant advantage in modern
medical care. One can also establish that decreased morbidity is available through diagnostics and
treatments by laser systems. So, will there be approval to use these lasers for more laser medicine and
surgery in this current cost conscious era? Can we now afford all this laser research and development?
A consortium in laser biomedical manufacturing, basic controlled research, and a comprehensive bio-
medical engineering program for instrumentation for what the patient needs — can we pay for all of
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this? Hopefully, the laser market of the future will continue to be developed and to enable the American
market to compete with the Japanese and European markets.
Another economic facet is the increased, often practical value in the training of physician assistants.
For example, this is done in the naval medical program for training of dermatology corpsmen as laser
technicians. This makes available a very effective dermatologist’s assistant. This training is offered mostly
in California at present.
Current biomedical lasers are becoming more flexible, smaller and perhaps not as expensive. There
are now more laser office and ambulatory surgical practices than ever before. Diagnostic laser facilities
will be more available in the coming years. As technology expands, the biomedical laser of the future
will include more multi-wave systems with diodes, heavy metal vapors and optical parametric oscillator
types. Adequate funding, because of the need for such lasers in laser biomedical engineering, will be
necessary, as well as the development of complex laser safety programs.
If the laser can continue to prove that it will reduce the cost of medical care and be used by trained
critical practitioners, in a country where laser medicine and surgery began, laser medicine and surgery
can continue as major parts of the practice of both medicine and surgery.
Leon Goldman, M.D.

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Preface
The purpose of assembling this book, with chapters from a group of experts whose backgrounds come
mainly from the experimental side of the physical sciences, was to try to set down the the basics of laser
interaction with tissue and describe how these basics have been applied in some of the medical specialties.
The reasoning behind this is that 1) there are many areas in medical science where lasers or modern
optics might have application if only some physical scientists knew about them and could transfer their
knowledge to the medical area, and 2) clinicians need to know some basic principles from physical science
to understand the opportunities and limitations of lasers and optical science as they try to apply these
devices in medicine.
Lasers have made many advances in medicine, especially in ophthalmology, dermatology and cardi-
ology. A wave of enthusiasm follows the use of lasers that causes patients to request its use, even when
it may not be warranted. Hopefully, this book will be able to convey to physicians enough basic infor-
mation to better assess a laser’s usefulness for a specific purpose, so they will not purchase or try to use
a laser when it is not the best solution.
The converse of enabling physicians to understand the limitations of a laser is to encourage them to
try using a laser when it may do a better job than the conventional practice. In this regard, I am pleased
and fortunate to have a foreword by the late Leon Goldman, known widely as the father of laser medicine.
Leon was a pioneering soul whose enthusiasm for laser use was instrumental in much of the early progress
in laser medicine. He was largely responsible for the founding of the American Society for Laser Surgery
and Medicine, which provides a forum for training and education and discussions among laser medical
professionals. I feel fortunate to have known Leon, and treasure his foreword to this book.
I am grateful to all the contributing authors for their hard work with their chapters and for their
encouragement. I am also grateful to George Pettit for his assistance in the early stages of the book, and
to Marcia Patchan for her help in keeping things organized at the finish.
Ronald W. Waynant
Clarksville, MD
Editor-in-Chief
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Editor
Ronald W. Waynant received his B.E.S. in electrical engineering from the Johns Hopkins University in
1962 and M.S.E.E. and Ph.D. from Catholic University in Washington, D.C. in 1966 and 1971, respectively.
From 1962 to 1966, Dr. Waynant worked in development and applications of solid state lasers at
Westinghouse in Baltimore and continued part time in electrooptics and low light level television until
1969, when he joined the Naval Research Laboratory, where he carried out gas laser research that led to
the first vacuum ultraviolet laser — the hydrogen laser. This work in the vacuum ultraviolet provided
the spark that began serious x-ray laser research. Dr. Waynant also worked on the kinetics of rare gas
halide excimer lasers, waveguide excimer lasers, microwave excited excimer lasers, and in excimer laser
lithography. In the course of this work, he has published nearly 80 scientific papers and has given more
than 90 contributed and invited talks on his work.
In 1986, Dr. Waynant joined the Food and Drug Administration to assist in the development of a laser
surgery research program that includes studies of basic laser interaction with tissue, fiber optic delivery
of laser energy to target tissue, and concern for long-term effects that might follow laser surgery.
Dr. Waynant is a member of the American Physical Society and the Society of Photooptical Instru-
mentation Engineers. He is a Fellow of the Institute of Electrical and Electronics Engineers, the Optical
Society of America and the American Institute of Medical and Biological Engineers.
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Contributors
Thomas P. Coohill George H. Pettit Brian C. Wilson

Siena College Summit Autonomous Ontario Cancer Institute
Loudonville, New York Orlando, Florida Princess Margaret Hospital
and
Craig Gardner Qiushi S. Ren University of Toronto
Rio Grande Medical Bascom Palmer Eye Institute Toronto, Ontario
Technologies, Inc. Biophysics Lab Canada
Albuquerque, New Mexico Miami, Florida
Mark H. Wholey
Jordan D. Haller Jonah G. Sinowitz Department of Radiology
School of Public Health Department of History Shadyside Hospital Pittsburgh
Columbia University Stanford University and
New York, New York Stanford, Connecticut University of Pittsburgh School of
Medicine
Tiina Karu Sune Svanberg Pittsburgh, Pennsylvania
Laser Technology Research Center Department of Physics
Russian Academy of Science Lund Institute of Technology
Moscow Region, Russian Federation Lund, Sweden Douglas R. Wyman
Department of Medical Physics
Stuart L. Marcus Keith P. Thompson Hamilton Regional Cancer Center
Dusa Pharmaceuticals, Inc. Department of Ophthalmology and
Valhalla, New York Emory University School of McMaster University
Medicine Hamilton, Ontario
Glenn N. Merberg Atlanta, Georgia Canada
Integration Services Group, Inc.
Bethesda, MD Ronald W. Waynant Osvaldo Yano
Food and Drug Administration College of Physicians and Surgeons
Mehmet Oz Center for Devices and Columbia University
College of Physicians and Surgeons Radiological Health New York, New York
Columbia University Rockville, Maryland
New York, New York
A.J. Welch
Jean-Marie Parel Department of Biomedical
Bascom Palmer Eye Institute Engineering
Biophysics Lab University of Texas
Miami, Florida Austin, Texas
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Table of Contents
1 Basics of Lasers
Ronald W. Waynant and Glenn N. Merberg
1.1 Laser Principles ........................................................................................................................... 1
1.2 Laser Materials ............................................................................................................................ 1
1.3 Pump Sources.............................................................................................................................. 3
1.4 Resonators ................................................................................................................................... 6
1.5 Major Types of Lasers ................................................................................................................. 6
1.6 Medical Lasers ........................................................................................................................... 14
1.7 Measuring Laser Power............................................................................................................. 15
1.8 Focusing Laser Energy .............................................................................................................. 15
1.9 Basics of Fiber Optics................................................................................................................ 16
1.10 Optical Materials....................................................................................................................... 20
1.11 The Future of Medical Lasers and Fiber Optics ...................................................................... 24
References ............................................................................................................................................ 25
2 Optical and Thermal Response of Tissue to Laser Radiation

A.J. Welch and Craig Gardner
2.1 Introduction .............................................................................................................................. 27
2.2 The Optical Response Of Tissue .............................................................................................. 28
2.3 Thermal Response..................................................................................................................... 41
2.4 Summary ................................................................................................................................... 45
References ............................................................................................................................................ 45
3 Dosimetry and Thermal Monitoring

Brian C. Wilson and Douglas R. Wyman
3.1 Introduction .............................................................................................................................. 47
3.2 Optical Dosimetry..................................................................................................................... 47
3.3 Thermal Dosimetry................................................................................................................... 68
3.4 Radiologic Imaging Methods ................................................................................................... 79
3.5 Summary ................................................................................................................................... 79
References ............................................................................................................................................ 80
4 Uses and Effects of Ultraviolet Radiation on Cells and Tissues

Thomas P. Coohill
4.1 Introduction to Ultraviolet Radiation ..................................................................................... 86
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4.2. A Division of the Ultraviolet for Photobiological Studies ...................................................... 86

4.3 UV Sources ................................................................................................................................ 88
4.4 Absorption of Ultraviolet ......................................................................................................... 90
4.5 Direct vs. Indirect Effects of UV............................................................................................... 96
4.6 Action Spectroscopy: Effect as a Function of λ. ...................................................................... 96
4.7 Effects of UV on Cells ............................................................................................................. 100
4.8 Complex Responses of Tissues............................................................................................... 101
4.9 Repair....................................................................................................................................... 103
4.10 General Effects......................................................................................................................... 104
4.11 A Perfect Laser for UV Photobiological Studies.................................................................... 104
4.12 Useful Review Texts ................................................................................................................ 105
References .......................................................................................................................................... 105
5 The Physics of Ultraviolet Laser Ablation

George H. Pettit
5.1 Introduction ............................................................................................................................ 109
5.2 Deposition of Ultraviolet Radiation in Organic Materials ................................................... 113
5.2 Target Decomposition ............................................................................................................ 120
5.3 The Ablation Plume ................................................................................................................ 122
5.4 Repetitive Irradiation.............................................................................................................. 128
References .......................................................................................................................................... 129
6 Tissue Diagnostics Using Lasers

Sune Svanberg
6.1 Introduction ............................................................................................................................ 135
6.2 Light Interaction with Tissue ................................................................................................. 136
6.3 Spectroscopic Diagnostics of Malignant Tumors ................................................................. 141
6.4 Spectroscopic Diagnostics of Atherosclerotic Plaque ........................................................... 151
6.5 Light Scattering and Tissue Transillumination ..................................................................... 157
6.6 Outlook.................................................................................................................................... 163
Acknowledgments ............................................................................................................................. 164
References .......................................................................................................................................... 165
7 Low-Power Laser Effects

Tiina Karu
7.1 Introduction ............................................................................................................................ 171
7.2 Primary and Secondary Mechanisms of the Action of Monochromatic Visible
and Near Infrared Radiation on Cells.................................................................................... 172
7.3 Explanation of Controversies and Limitations of Low–Power Laser Effects
on Cellular Level ..................................................................................................................... 187
7.4 Clinical Applications of Low-Power Laser Effects ................................................................ 196
7.5 Summary ................................................................................................................................. 200
References .......................................................................................................................................... 201
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8 Therapeutic and Diagnostic Application of Lasers in Ophthalmology

Keith P. Thompson, Qiushi S. Ren, and Jean-Marie Parel,
8.1 Introduction ............................................................................................................................ 211
8.2 Basic Ocular Anatomy and Physiology.................................................................................. 212
8.3 Transmission and Absorptive Properties of Ocular Tissues................................................. 214
8.4 Photothermal Laser Applications........................................................................................... 217
8.5 Photodisruptive Laser Applications....................................................................................... 231
8.6 Photochemical Laser Applications: Photoablation and Photodynamic Therapy................ 233
8.7 Diagnostic Laser Applications ................................................................................................ 239
8.8 Summary ................................................................................................................................. 240
Acknowledgments ............................................................................................................................. 241
References .......................................................................................................................................... 241
9 Cardiovascular Applications of Lasers

Jordan D. Haller
9.1 Introduction ............................................................................................................................ 247
9.2 History (with Jonah G. Sinowitz) ........................................................................................... 248
9.3 Anatomy and Pathology ......................................................................................................... 249
9.4 Physics...................................................................................................................................... 251
9.5 Angioplasty (with Mark H. Wholey) ...................................................................................... 262
9.6 Transmyocardial Laser Revascularization (TMLR) .............................................................. 268
9.7 Other Surgical Applications of Lasers in Cardiology
(with Osvaldo J. Yano and Mehmet C. Oz)............................................................................. 271
9.8 Aids to Welding: Tissue Solders ............................................................................................. 272
References .......................................................................................................................................... 278
10 Lasers in Photodynamic Therapy

Stuart L. Marcus
10.1 Introduction ............................................................................................................................ 287
10.2 Tissue Optical Properties, Photobleaching, and Light Delivery Systems............................. 289
10.3 PHOTOFRIN PDT for Superficial Bladder Cancer .............................................................. 291
10.4 PHOTOFRIN PDT in the Treatment of Endobronchial Lung Cancer................................ 296
10.5 PHOTOFRIN PDT in Gynecologic Malignancies ................................................................ 300
10.6. PHOTOFRIN PDT in Head and Neck Cancer...................................................................... 302
10.7 PHOTOFRIN PDT in Gastrointestinal Cancer..................................................................... 304
10.8 PHOTOFRIN PDT for Intraoperative Abdominal or Thoracic PDT.................................. 306
10.9 PHOTOFRIN for Intraoperative PDT in the Treatment of Intracranial Tumors .............. 307
10.10 PHOTOFRIN PDT in the Treatment of Ocular Cancer....................................................... 310
10.11 PHOTOFRIN PDT in the Treatment of Cutaneous and Subcutaneous Tumors............... 311
10.12 New Photosensitizers for PDT ............................................................................................... 313
10.13 Conclusions ............................................................................................................................. 317
References .......................................................................................................................................... 317
Index...................................................................................................................................................325
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1
Basics of Lasers
1.1 Laser Principles....................................................................... 1

1.2 Laser Materials........................................................................ 1
1.3 Pump Sources......................................................................... 3
1.4 Resonators............................................................................... 6
1.5 Major Types of Lasers ............................................................ 6
1.6 Medical Lasers ...................................................................... 14
1.7 Measuring Laser Power........................................................ 15
Ronald W. Waynant 1.8 Focusing Laser Energy ......................................................... 15
Food and Drug Administration 1.9 Basics of Fiber Optics .......................................................... 16
Center for Devices and General Considerations
Radiological Health
1.10 Optical Materials .................................................................. 20
Glenn N. Merberg 1.11 The Future of Medical Lasers and Fiber Optics ................ 24
Integration Services Group, Inc. References ........................................................................................ 25
1.1 Laser Principles

To understand the operation of the laser requires a knowledge of the energy levels associated with atoms,
ions and molecules. In thermal equilibrium, the energy levels are populated according to the Boltzmann
distribution, which forbids the conditions in which an upper level might have a greater population than
a lower level. Because, for lasing, an upper level must be more highly populated than the lower level,
lasing will not take place. Lasing can take place only when a material is not in thermal equilibrium. This
non-equilibrium is created by an excitation source sometimes called a “pump” source. Just as thousands
of atoms, ions or molecules can be laser materials, numerous pump sources can excite the materials.
In many cases, the gain produced by the pumped laser material is low. To make a device, it is necessary
to use an optical resonator to repeatedly reflect the signal through the amplifying material to add to the
intensity. Therefore, the basic elements of a laser are: (1) a laser material, (2) a pump source and (3) a
resonant cavity, as shown in Figure 1.1. Figure 1.2 shows the concept of an excited material in which the
higher energy level is more populated than the lower energy level. This is clearly a non-equilibrium
system. Without the resonator, the excited system is capable of amplifying a signal only once, as shown
in Figure 1.3. The addition of a resonator (two mirrors aligned to precisely reflect the energy back and
forth) allows a signal to be amplified to high intensity. Figure 1.4 shows the laser oscillator that results.
1.2 Laser Materials

Laser materials have been the focus of 30 years of research. (A list of generic laser materials is contained
in Table 1.1.) The research began with solid state lasers such as ruby and neodymium doped glass
lasers. In the early days of lasers, all the “easy-to-grow” solids were grown with a wide collection of
dopants. Most of these devices were inefficiently pumped by flashlamps and many of the materials
0-8493-1146-2/02/$0.00+$1.50
© 2002 by CRC Press LLC 1
2 Lasers in Medicine
feedback and mirror

mirror oscillation atmos (laser medium)
laser output
beam
R=100% R=80%
pumping process
FIGURE 1.1 Basic laser components.1
energy
upper
level
laser
action population
inversion
lower
level
population
FIGURE 1.2 Population inversion.1
Inverted laser medium
Input amplified output

light beam light beam
FIGURE 1.3 Laser Amplifier.1

Basics of Lasers 3
reflected
light waves
Inverted laser medium
100% partially
reflecting transmitting
mirror mirror
FIGURE 1.4 Laser oscillator.1
TABLE 1.1 Laser Materials2

Laser Material Comments
1. Solid
a. Doped Crystal Host Ruby, garnets, etc., usually optically pumped
b. Semiconductor Electrically pumped, high efficiency
2. Liquid
a. Dyes in Solvent Tunable, optically pumped
3. Gases
a. Atomic Rare gases, metal vapor
b. Molecular Infrared, ultraviolet
c. Excimer
- Diatomic Ultraviolet
- Triatomic Visible
- Ionic Ultraviolet, vacuum ultraviolet, x-ray
were easily damaged by the radiation they generated. These problems fueled the development of gas
lasers, which were rf- or dc-discharge pumped in the beginning. Semiconductor lasers were discovered
but made little impact because of their need for cryogenic cooling. The desire for more energy and
more laser types led to flowing gas lasers and to high energy electron beams for laser excitation.
Electron beams worked just fine for laser research, but they were impractical for most applications.
Low inductance discharge excitation systems were developed for excimer lasers. In the meantime, the
development of the heterostructure semiconductor enabled room temperature operation of diode
lasers, which have high energy conversion efficiency. Combinations of diode arrays made suitable
excitation sources for solid materials, which led to a renaissance of solid state laser research and a
desire to retry many of the old materials with the new pump. The combination has led to smaller,
more practical lasers capable of portable uses.
From this more than 30 years of laser research has come a handful of lasers that have begun to be
developed for medical applications. We will expand on these lasers below. Here, it is sufficient to say
that much must be done to make these devices better and more appropriate for surgery and other
medical applications.
1.3 Pump Sources

The first laser was pumped by a flashlamp. Flashlamps of the sort appropriate for laser pumping were
developed by Edgerton for flash photography. They consist of a quartz envelop, two tungsten elec-
trodes, a low pressure gas — usually xenon — and an energy storage system suitable for single or
repetitive pulsing of the lamp. A collection of some of the common flashlamps that have been used
is shown in Figure 1.5. The lamp emits a blackbody energy distribution of temperature near 5500 K.
The dopant in the laser material absorbs a small fraction of this energy, hence the poor efficiency
from flashlamp pumping.
FIGURE 1.5 Typical linear and helical flashlamps used for laser pumping.2
Beam
Output
mirror
Total
reflector
FIGURE 1.6 DC discharge system.
RF
supply
Metal-rf
electrodes
Waveguide
Ceramic insulator
FIGURE 1.7 RF discharge excitation.
Discharge pumping allows the energy source to drive the exciting electrons directly within the laser
material. There is no conversion to optical energy and back to excited state as in flashlamp pumping,
but discharge pumping has its problems too. Greater care must be taken of electron temperature to
optimize excitation of the correct level. Gases can be added to the discharge to improve energy transfer
to the excited state or to remove energy from the lower laser level, but the effects of these gases on the
discharge conditions must be a concern. Figure 1.6 shows a typical longitudinal dc discharge, while
Basics of Lasers 5
MIRROR
OPTICAL WINDOW
CATHODE (SCREEN)
ELECTRON BEAM
-HV
(ELECTRON BEAM + HV (DISCHARGE
POWER SUPPLY) POWER SUPPLY)
ANODE (SOLID)
DISCHARGE
REGION
FIGURE 1.8 E-beam system for laser excitation.
Figure 1.7 is an rf discharge. Both types of discharge have been used frequently for excitation of low
pressure gas lasers.
Electron beams are capable of exciting nearly any material. Solids and semiconductors have been studied
by e-beam excitation as well as gases. High pressure gases can be excited by e-beams quite easily. E-beams
are excellent for quick studies, but are impractical for lasers that are intended for applications. The
maintenance required by electron beam machines is too costly for e-beam pumped lasers to be useful.
A typical electron beam pumped laser system is shown schematically in Figure 1.8.
Electric current from rather low voltage sources can be used to pump semiconductor lasers. Since
this type of pumping is at least 30% efficient (compared with a few percent at best from most other
sources), it is easy to see the widespread popularity of diode lasers and the interest in using them to
pump other lasers.
Chemical excitation is, in some ways, an attractive and efficient process. By inducing a chemical
reaction that leaves a reactant in an excited state, it is possible to build a population inversion
capable of laser action. Very little energy is needed from the outside to ignite the chemical reaction.
Since the energy is limited only by the volume of the population, these devices are scalable to high
power. (A list of numerous laser-producing reactions is given in Table 1.2.) However, many of the
gases are toxic or highly reactive and handling a large volume of them is an unpleasant, unsafe and
costly task.
Nuclear pumping has some attraction because the emission from nuclear reactors can excite laser
materials, but this method of pumping is impractical for lasers that must be used for applications remote
from the reactors.
TABLE 1.2 Major Chemical Lasers2

Laser Typical Reaction Wavelength (nm)
I O2* + I → O2 + I* l.3
HF (overtone) Same as HF 1.3–1.4
HF F + H2 → HF* + H 2.6–3.5
H + F2 → HF* + F
HCl H + Cl2 → HCl* + Cl 3.5–4.1
DF F + D2 → DF* + D 3.5–4.1
D + F2 → DF* + D
HBr H + Br2 → HBr* + Br 4.0–4.7
CO CS + O → CO* + S 4.9–5.8
CO2 DF* + CO2 → CO2* + DF 10–11
1.4 Resonators
Two types of resonators can be important for laser applications: stable and unstable. Each type has some
important principles.
Stable resonators are the most common type. Examples of numerous stable resonators, which have
been given considerable research, are shown in Figure 1.9. The most popular are characterized by a
repeated and stable radiation pattern within the resonator. Stable resonators are characterized by some-
what wider beam divergence and somewhat poorer coupling to the excited gas volume inside the tube.
These resonators are needed to achieve oscillation in low gain systems.
Unstable resonators are practical only with high gain laser materials, but they produce just the opposite
effects to stable resonators. Examples are given in Figure 1.10. These lasers do not direct a ray back and
forth over the same path, but actually utilize a mild walk-off path that couples much of the excited
volume into an output that can be made nearly parallel. This non-divergent beam can be focused into
a smaller focal spot than the stable resonator output.
1.5 Major Types of Lasers

Far Infrared lasers
1. CO2 (10.6 µm): The carbon dioxide laser sparked a recovery of lasers for surgery after early
difficulties with pulsed laser treatment of tumors. Figure 1.11 shows the energy levels of the CO2
laser. Carbon dioxide lasers are usually discharge excited. High CW powers are possible with either
dc or rf excitation of mixtures of gases that include CO2, N2, He and H2 in approximately a
1:1:8:trace ratio. Examples of these excitation systems that could be used to excite CO2 are shown
in Figures 1.6 and 1.7. Powers of 50–100 W can easily be obtained from a modest 1–4 m tube.
Mid Infrared
1. Er:YAG (2.94 µm): This is a vibronic solid state laser with an emission line exactly matching the
region of highest absorption of water. The crystal is not as durable as some of the other materials.
It can be run at a 5–10 Hz rate with average power to 10–15 W. The system can be q-switched
with some difficulty. Ordinary stochastic pulsing can be obtained for periods of a few hundred
microseconds. The total energy per pulse can be as high as 0.5 to 1.0 joules per pulse, and it can
operate at a rate of 1–6 pps.
2. Ho:YAG (2.1 µm): Holmium is another vibronic laser with emission near 2 microns. It also can
be used with the garnet hosts and, in some cases, thulium is used as a sensitizer (i.e., the thulium
absorption bands extract energy from the pump, but transfer it to holmium to improve its
efficiency. Thulium lasers as well at 2.9 µm. Holmium suffers from room temperature population
of the lower laser level, which will make it difficult to operate without cryogenic cooling.
3. Nd:YAG (1.06 µm): One of the early lasers and still a most important one, it encounters little
attenuation and penetrates tissue rather well — from 1–5 mm or so. Figure 1.12 gives the energy
levels of the Nd:YAG laser. The laser can operate cw or pulsed with pulses from nanoseconds to
picoseconds and pulse compression to femtoseconds is possible. The laser can be frequency
doubled, tripled, quadrupled routinely and even higher harmonics can be generated. Numerous
garnets and other hosts can be used with neodymium to make slightly different wavelengths.
The device can be diode pumped or flashlamp pumped. A wide variety of pumping configu-
rations have been used. Typical output powers for CW and pulsed operation are given in the
Table 1.3.
Near Infrared
1. Alexandrite (700–826 nm): This material is Cr:BeAl2O4 and its energy levels are not very different
from those of ruby, as shown in Figure 1.13. It was the first solid state tunable laser and is one of
Basics of Lasers 7
R1 (Mirror radius) = (a) Plane-parallel R2 =
8
L
R2 > (b) Large-radius mirrors >> L
R1 = L (c) Confocal R2 = L
a/ 2
R1 = L / 2 (d) Spherical R2 = L / 2
R1 > L (e) Concave-convex R2 = - (R1 - L )
R1 = L R2 =
8
(f) Hemispherical
FIGURE 1.9 Examples of

stable resonators.
negative branch confocal
positive branch confocal
FIGURE 1.10 Examples of unstable resonators.

CO2 N2 He
C
O C O O O O C O N N He
C
Symmetric stretch Bending Asymmetric stretch
J(odd)
3000 55
53
0 3 v=1
00 1 1
J(even)
µm Next quantum state in
.4 µ
m J
2000 58
10 9.4 helium is 67.7 times the
56 54
Energy (cm-1)
52 v = 0 to v = 1 spacing
2 in nitrogen
0 2
0
10 0 0 0
02 0
1000
4.23 µm
01'0
0
00 0 v=0
0
v1 v2 v3 1'S
FIGURE 1.11 Energy levels of the CO2 molecule.3
20 Pump
bands
18
16
14 4 11502 cm-1 R2
F3/2 11414 R1
4
Laser
12 F3/2
Energy ( x 10 cm )
transition
-1
3
10 4
I 15/2 ~ 6000 cm-1
Laser
transition
8
4
I 13/2 ~ 4000 cm-1
4
I 15/2
6
2526
4 2473
I 13/2 4
4 I 11/12 2146
2111
4 2029
I 11/2 2001
2
4 848
I 9/2 Ground 4 311
0 I 9/2 197
level
0 134
FIGURE 1.12 Energy levels of the Nd: YAG laser.3

Basics of Lasers 9
TABLE 1.3 Neodymium Laser Performance2

Type Pump Wavelength (nm) Power (W)
Pulsed quad Nd glass Flashlamp 263–266 0.04

Pulsed quad Nd:YAG Flashlamp 266 0.001–2
Pulsed trip Nd:YAG Flashlamp 355 0.001–20
Pulsed doub Nd:YLF Diode 523 0.00002-0.01
Pulsed doub Nd:YLF Flashlamp 523–527 1–3
CW doub Nd:YAG Diode 532 0.001–0.08
Pulsed doub Nd:YAG Diode 532 0.000001–0.0004
Pulsed doub Nd:YAG Flashlamp 532 0.01–50
CW Nd-YLF Diode 1047 0.035–0.25
CW Nd-YLF Arc lamp 1053 20
CW ND, Cr-GSGG Diode 1061 0.1
CW Nd-YAG Diode 1064 0.002–3
CW Nd-YAG Arc lamp 1064 0.5–1800
Pulsed Nd-YAG Flashlamp 1064 1500
25 1
4 4 T2
T1 T1
2 2
T2 Vibronic T2 4
T1
20 band
4
T2
Tunable wavelength
4
Energy, 103 cm-1
T2
Fixed wavelength
2 2
T1 T1 1 4
15 2 E A2
E 2
E
3
T1
10
0.701
0.6943 0.6804 -0.818 3 4
µm µm
T2 T2
5 1.63- 2.11
1.61- 1.74
µm µm
4 4 3 4
0 A2 A2 A2 T1
3+ 3+ 2+ 2+
Cr Cr Ni Co
(Al2 O3) (BeAl2O4) (MgF2) (MgF2)
Ruby Alexandrite
FIGURE 1.13 Energy levels of several vibronic lasers.2
a number of vibronic solid state lasers listed in Table 1.4. While the laser suffers from a lower
stimulated emission cross section than neodymium, the cross section improves as the crystal
temperature rises.
Visible
1. Ti:Al2O3 (660–1180 nm): Titanium doped sapphire has a large cross section comparable to neody-
mium, but its excited state lifetime is only 3.2 µs, too short to store and build up a substantial
upper laser level population. Laser pumping is usually required.
2. Cr: Al2O3 (ruby–694.3 nm): The first laser produced, its high threshold makes it less desirable that
many of the other lasers. Still, it finds a few applications where its wavelength and pulsed charac-
teristics are desirable, such as in tattoo removal.
3. Semiconductor Diode Lasers (635–1550 nm): Semiconductor lasers were first produced in the
early 1960s. Initially GaAs devices, they were quite weak in output and required cryogenic cooling.
TABLE 1.4 Vibronic Lasers

Wavelength
Type Pump source Operation (nm)
Alexandrite Arc lamp CW 730–810

Alexandrite Flashlamp Pulsed 701–858
Ce-YLF KrF Excimer Pulsed 309–325
Co-MgF2 1320 nm Nd:YAG Pulsed 1750–2500
Cr-LiCaAlF6 Laser or lamp Pulsed or cw 720–840
Cr-LiSrAlF6 Laser or lamp Pulsed or cw 760–920
Emerald (Cr doped) Laser Pulsed or cw 720–842
Fosterite (Cr doped) Laser Pulsed or cw 1167–1345
Thulium-YAG Laser CW 1870–2160
Ti-Sapphire Usually laser Pulsed or cw 660–1180
10 µm
250 µ
m
p θ1
n
θ11
Active
region
Facet
FIGURE 1.14 Diode laser structures.
A schematic of the tiny structure of these diode lasers is shown in Figure 1.14. Improvements over
the years have developed new heterostructure junctions that allow room temperature operation.
Output power of arrays are now powerful enough to be used as pumps for other materials. These
devices have always been efficient emitters, and thus make a convenient, low voltage pump espe-
cially useful with solid state laser materials. These devices are usable in their own right as easily
modulated sources for fiber optic communication sources. The new technology has allowed these
devices to move to both shorter and longer wavelengths as well. The wavelengths available and
the materials that emit them are given in Table 1.5.
Table 1.5 shows the power available from diode lasers at the various wavelengths. Pumping of
lasers can be accomplished by the arrays that can be constructed at 810 nm. This wavelength is
absorbed strongly by some solid state materials.
For communications purposes, a diode composed of In0.73Ga0.27As0.58P0.42 will produce a
diode emission of 1.33 µm, and a material composed of In0.58Ga0.42As0.9P0.1 will emit at 1.55µm.
These wavelengths are preferred for communications because of the improved transmission of
fiber optics.
4. Copper/Gold Vapor (511, 578/628 nm): Metal vapor lasers such as those operating on copper and
gold can achieve high average power by running at high pulse repetition rates. Unable to run
Basics of Lasers 11
TABLE 1.5 Semiconductor Laser Performance

Wavelength (nm) Material Power
635 InGaAsP 3 mW
660 InGaAsP 3 mW
670 Ga0.5In0.5P 10 mW
750 GaAlAs 8 mW
780 GaAlAs 35 mW
810 GaAlAs 100 mW single
810 GaAlAs 10 W linear array
810 GaAlAs 60W quasi-cw (pulsed)array
810 GaAlAs 1500W quasi-cw stacked array
830 GaAlAs 150 mW
850 GaAlAs 100 mW
880 or 895 GaAlAs Pulsed only
905 GaAs Pulsed only
910 InGaAs Pulsed only
980 InGaAs 50 mW
continuously because of lower level bottlenecking, these lasers can operate at a pulse rate of several
kilohertz and can reach an average power of more than 100W. Gold lasers are of considerable
medical interest because the wavelength is appropriate for photodynamic therapy. Other metal
vapor lasers are shown in Table 1.6.
A second problem of metal vapor lasers is that no good source of blue radiation has been found
to allow red, green, or blue color projection. Several candidates have been worked on, but are not
able to compete with the strength of the lasers shown in Table 1.6.
5. Dye Lasers (broadly tunable): Dye lasers are perhaps the least understood, but some of the most
widely applied lasers. Originally made from some of the wide variety of dyes used by the clothing
industry, little is known about the energy structure of these large molecules. Their absorption and
emission spectra usually show broad features. Typical generic energy levels are shown in
Figure 1.15. The upper state lifetimes are short and require fast pumping pulses (nanoseconds.
Pumping is usually accomplished by flashlamp or by laser excitation. The broad emission enables
tunable laser emission to be generated, which allows spectroscopic studies. In addition, the wide
bandwidth also allows ultrashort pulse generation.
Dyes are mixed with a wide variety of solvents at concentration levels of millimoles or lower.
For continuous or rapidly pulsed operation, and to prevent deterioration by the short wavelength
pump, the dyes are rapidly circulated to lower temperature. A selection of dyes, solvents and
pumping sources that allow the generation of laser wavelengths from the ultraviolet to the infrared
and with power up to a few watts average has been produced. Several schemes have been invented
to allow tunable operation of dye lasers. A rather simple method is shown in Figure 1.16.
Figure 1.17 shows a more sophisticated ring dye laser.
6. Argon/Krypton (515, 488/647): These ion lasers typically emit tens of watts and have been built
to emit hundreds of watts of cw laser power. They are gas lasers operating at about a torr of
pressure of pure argon or krypton. However, their construction has been a monument to gas tube
design. Figure 1.18 shows the energy levels involved in the argon ion laser. Since energy injected
into the gas must first ionize the gas and then excite and invert the upper ion level, a high current
is necessary. The lasers are very inefficient and operate at high power only when pumped with
high discharge current. The high current discharge places such thermal stress on the tube that
only a few materials can survive even when a magnetic field is used to keep the discharge away
from the walls, and strong water cooling is used to remove heat.
The high power continuous emission of these devices offers blue or green emission capable of
driving some chemical reactions. When mode locked to generate a few tens of picosecond pulses,
TABLE 1.6 Metal Vapor Lasers

Wavelength Power
Element (nm) (relative to Cu) Comments
Copper 511, 578 1

Gold 628 0.1 to 0.3
312 Low Secondary laser line
Barium 1130 Low Ba liquid a problem
1500 0.3 to 0.5 Ba liquid a problem
Lead 722.9 0.2 to 0.3 1000-1100 °C temperature
Manganese 534 0.2 to 0.3 Mn vapor a problem
1290 Mn vapor a problem
Calcium 852.4
866.2
Triplet absorption
(can soak up laser
emission)
Excited state (S1) Radiationless
decay
Excitation
Laser Alternative path through

emission triplet states (not a laser
transition)
Ground state (S0) Radiationless

decay
Singlet Triplet
states states
FIGURE 1.15 Typical dye laser energy levels.2
the laser can be used as a synchronous pump for dye lasers. When operated at lower pressure, the
generation of higher stages of ionization can produce shorter cw wavelengths — some reaching
the mid-ultraviolet nearly to the vacuum ultraviolet.
Ultraviolet
1. Excimer Lasers: The name excimer comes from two words, excited and dimer. A dimer is a molecule
made up of two similar atoms. Some dimers form only when one of the atoms is excited. An
example is the rare gases that form an excited dimer or excimer such as Xe2, which lives as an
excimer for less than 200 ns. While numerous rare gas excimers can be formed at high pressure
Basics of Lasers 13
Cavity focusing mirror

Pump beam
Prism (fixed)
Focusing lens
Dye cell or jet
Output mirror
Rear cavity mirror
(moved to tune
wavelength)
Output beam
FIGURE 1.16 Tunable dye laser.2
Ion laser pump beam

Pump mirror
Astigmatism
Dye jet compensator
Mirror Mirror
Variable focus
auxiliary beam waist
Double galvoplate
Output Output
mirror beam
Mirror
Unidirectional Thin Scanning Birefringent Collimated arm

device etalon etalon filter
FIGURE 1.17 Ring dye laser.
and pumped with electron beams, it has been found that rare gases form molecules with halogen
atoms much more easily. These rare gas halogen “excimers” can be formed at only an atmosphere
or two and can be pumped with low inductance discharge systems.
Because the upper laser level has such a short lifetime, excimer laser pulses are usually 10–20
ns in duration. Typical pulse energies range from 100 to 500mJ from modest excimer lasers.
Repetition rates of 50 to 100 Hz or greater also are typical. As this presents a high power to fibers
used to transmit the radiation, efforts have been made to lengthen the pulse to 100–200 ns.
Although silica windows transmit to short wavelength, no silica fiber has been satisfactory for
delivery of ArF (193nm) radiation. In addition, the use of halogen gases poses a safety problem
during the refilling and gas changing cycles. Table 1.7 shows the gas combinations and the wave-
lengths they generate.
Considerable interest has been generated by improvements in the F2 laser at 157 nm brought
on by the use of He in the discharge. This system, technically not an excimer, is now capable of
more than 100 mJ per pulse and can be used as a pump for other solids or gases.
2. Free Electron Lasers (Tunable): Free Electron Lasers (FELs) are discussed here only because of the
interest in these devices for medical applications. The FEL operates on the principle of perturbing
a beam made up of free electrons by use of a series of magnetic pole pieces arranged in a section
of a storage ring. This magnetic pole assembly is called a wiggler. FELs are large devices that
3/2 4p 2p0
4p 2D0 3/2 1/2
1/2
5/2 3/2
5/2 4p 4D0
7/2
4965 5145
4545
150,000 o
4765
4880 A 4658 5287
4889
140,000 1/2
4s 2p
Energy (cm-1)
3/2
130,000
+
Ar 3p5 2p 15.75 V
0 + 1.P
> 50 laser transitions in 127.109.9 cm-1
Arl, λ 0.7 30 µm
3p6 'S
0 Ar
FIGURE 1.18 Energy levels of argon ion laser.
TABLE 1.7 Rare Gas Excimer Lasers (Bold indicates strongest)

F Cl Br I
Xe 351, 353 308 282 253

Kr 249 222 206
Ar 193 175
Ne 108
currently exist as facilities at a few locations. As facilities they offer tunable, directional laser emission
having many of the properties of the more conventional lasers. Currently operating systems emit
mostly in the mid infrared from about 2–10 microns and have macropulse (1–2 microsecond pulses)
energy of about 1–200 mJ at a rate of 10–30 Hz. Within the macropulse is a series of pulses 2 ps
wide separated by 350 ps. Currently, these devices at Duke University, the University of California
at Santa Barbara (200–400 µm wavelength), Stanford University and Vanderbilt University are being
used to conduct exploratory research on numerous medical problems. A new storage ring FEL
designed to work at shorter wavelengths (from the visible through the ultraviolet, vacuum ultraviolet
and perhaps into the x-ray region) has been built at Duke University.
1.6 Medical Lasers

All of the above lasers can and have been used for medical purposes. If other wavelengths are needed,
they can likely be found among lasers already discovered. In a sense, however, the problem of lasers in
medicine lies not in discovering a magical wavelength, but in learning how to best deliver the right
wavelength, the right pulse width, the right pulse shape, the right beam profile and the proper amount
of energy to create the best medical outcome possible. The exact shape of the box(es) that contains the
equipment will not be discussed in detail. That is a matter of choice for the medical device industry,
which, nonetheless, must produce a safe, effective and competitive laser system.
Basics of Lasers 15
1.7 Measuring Laser Power

Measurement of laser power is, in many cases, simply a matter of placing a power meter in the beam
path. Numerous companies make excellent meters capable of making accurate measurements of laser
power and energy. Of somewhat greater concern, however, is the measurement of the laser pulse width
and phenomena associated with time dependent change. This is no problem for pulses of nanosecond
width or greater because excellent electronic systems exist that can make accurate measurements. Below
a nanosecond, time dependent measurements become more difficult.
If the pulse wavelength is in the visible, streak tubes and image intensifiers are able to record the pulse.
Electronic devices as well have response down to 50 ps or so. However, if the time variant pulse is in the
infrared, the problem is somewhat different. Photon energies are not capable of being seen by traditional
photocathodes. Nonlinear correlation can give an approximation of the pulse shape, but these techniques
do not allow real time, pulse-to-pulse visualization.
1.8 Focusing Laser Energy

Focusing laser energy produces one of the most difficult to measure parameters — focal spot size. This
is difficult to measure because it usually results in an intensity that evokes a nonlinear or damaging
response on any material that may be placed at the spot. Exact methods of measuring spot size have not
been developed.
There are some guidelines that can be used, however. First, a more or less uniform phase output beam
of wavelength, λ, emitted through an aperture, d, will have an angular divergence, ∆θ, such that
λ
∆θ = --- (1.1)
d
When a lens of focal length, f, is placed in the above uniform beam, then the beam can be focused to
a spot size, do, where
d o = ( F# ) ( λ ) (1.2)
and
f
F# = -- (1.3)
d
and is known as the f-number.

One further approximation, as the depth of field is also f-number dependent, an estimate of the volume
in the focal region, Fv, is that
2
F v = ( F# ) ( λ ) (1.4)
These approximations can be of help when focusing a beam onto a target such as tissue or a fiber
surface. Since beams may not be perfectly uniform or diffraction limited, and lenses may have aber-
rations as well, it is useful to devise methods to get better information on focal spot size for each
specific application.
1.9 Basics of Fiber Optics
1.9.1 General Considerations

Fiber optics can be thought of as conduits for light. In fact, before the advent of modern fiber optics,
hollow tubes with reflective inner surfaces were used to guide light energy. Today’s fiber optics are
solid-core flexible strands of highly transparent material. Light is guided through the fibers by a
process called total internal reflection (TIR). High quality optical fibers can carry light for miles with
negligible attenuation.
TIR is a consequence of the refraction of light by a refractive index gradient. Refraction can be defined
by considering the interface between two optical materials with indices of refraction nl and n2. A ray of
light crossing the interface from nl to n2 is deflected by the index mismatch according to Snell’s law:
n1 sin θ1= n2 sin θ 2 (1.5)
where θ 1 and θ 2 are the angles the ray of light makes with the normal on either side of the interface. In
the case where n1 > n2 the light will be deflected away from the normal upon crossing the interface.
Figure 1.19 illustrates refraction for light rays with various angles of incidence on an interface with n1
> n2. Note that, for many incident rays, there are two resultant rays. These rays can be thought of as the
reflected and transmitted rays. A reflected ray always leaves the interface at an angle, θ r , equal to the
angle of incidence, θ i. Its intensity is a function of the index mismatch between the two materials and
the angle of incidence. The transmitted ray has a path defined by Snell’s law, as described above. Some
incident rays, which are said to be totally internally reflected, produce only reflected rays.
Light rays with angles of incidence greater than the critical angle, θ cr , experience TIR. The critical
angle, θ cr , is defined by
sin θ cr = n1/n 2 (1.6)
Fiber optics guide light by TIR. The fiber typically consists of concentric layers, as shown in Figure 1.20.
The core material is surrounded by a cladding material of slightly lower index of refraction. Light is
contained in the fiber by TIR at the core-cladding interface and is guided along the fiber.
Numerical Aperture
To be guided by the fiber optic, light must be launched so that it satisfies the criteria for TIR. Light
entering the fiber is refracted at the end face of the fiber, and must strike the cladding at an angle greater
transmitted ray
θ2
n2 interface
n1
θ1 θr θcr
reflected ray
FIGURE 1.19 Refraction and total internal reflection.

Basics of Lasers 17
Buffer coating
Core
Cladding
FIGURE 1.20 Geometry of a fiber optic.
acceptance cone
θac
FIGURE 1.21 Numerical aperture and acceptance cone.
than the critical angle. Using simple geometry, we can define a cone that includes all the rays that will
be guided by the fiber, as is shown in Figure 1.21. This cone is called the acceptance cone, and the sine
of its half-angle, θ ac, is defined as the Numerical Aperture (NA) of the fiber. The NA is related to the
indices of refraction of the core and cladding of the fiber by the equation
2 2
NA = sin θ ac = n core – n clad (1.7)
Fresnel Reflection
Light crossing a refractive index gradient will be partially reflected, as was discussed previously. Similarly,
light launched into a fiber will be partially reflected at each of the end faces. The equation for reflection
of light striking an interface at normal incidence is
2 1 2 2 1 2
(n – n ) + (k – k )
R = -----------------------------------------------------
2 1 2 2 1 2
- (1.8)
(n + n ) + (n + n )
where R is the fraction of the light that is reflected, n and k are the real and imaginary components of
the indices of refraction, respectively, and the subscripts 1 and 2 denote the media on either side of the
interface. The quantity k is often referred to as the extinction coefficient, and can be considered negligible
over spectral regions where media 1 and 2 are highly transparent. A simple approximation of the total
light transmitted through a fiber after reflections from both end-faces are considered is given by
T = 2n/n2 + 1 (1.9)
where T is the fraction transmitted and n is the index of refraction of the core of the fiber. Conventional
fiber optics have indices of refraction of about 1.5. Therefore, about 92% of the light launched into the
fiber can be transmitted. Sometimes, anti-reflective coatings are applied to the ends of fibers, but these
coatings are wavelength specific and need to be reapplied every time the fiber is cleaved.
Bending
Fiber optics can be bent gently without inducing substantial optical attenuation. Only at severe bends
does the optical transmission begin to fall. The severity of bending of a fiber is described in terms of
bend radius, which can be thought of as the radius of the circle the fiber would form if it were bent into
a closed loop. Figure 1.22 shows the effects of bending on the transmission of fibers with various NAs.5
In addition to optical considerations, fibers have mechanical minimum bend radii. Bending fibers to
smaller radii results in catastrophic failure. Conventional fibers, which are made of synthetic silica, are
extremely strong and, thus, very flexible. The recommended minimum bend radius for silica fibers is
about 50–100 times the fiber diameter.5 For long-term bends, such as in a rigid device or for packaging,
the suggested minimum bend radius is about twice this value.
Often, the flexibility of a fiber will be specified in terms of strain-to-failure. Strain is a unit of
deformation, and is defined as change in length per unit length, or
ε = ∆l/l (1.10)
where ε is strain, l is sample length and ∆ l is the elongation of the sample under some load. Strain-to-
failure defines the strain point at which the sample will fracture. The quantity, ε f , is related to the
minimum bend radius by the relationship
εf = r/R (1.11)
where r is the fiber radius and R is the minimum bend radius.
100
90
Transmission (%)
80
70
TECS (NA = 0.39)

PCS (NA = 0.40)
60 GLPC (NA = 0.18)
0 1 2 3 4 5 6
Bend Radius (cm)
FIGURE 1.22 Effect of bending on fiber transmission.

Basics of Lasers 19
Laser Damage
High energy lasers are capable of causing damage in the fiber optics used for their delivery. The sources
of this damage are widespread.6 For cw, or long pulsewidth lasers (>50 µ s), the damage is most frequently
associated with gradual heating of the fiber. Often, this is a result of misalignment of the laser. Typically,
the cladding material or epoxy in the connector heats and causes failure of the fiber. For short pulse
lasers, however, there is the added issue of high peak powers. Short pulses with high peak powers may
cause photoacoustic effects that damage the fiber. In addition, these lasers may cause a plasma to develop
at the launch face of the fiber. The plasma is witnessed as a blue spark at the end of the fiber accompanied
by an audible snapping sound. Most often, laser damage occurs on either of the end faces, or at the point
of first focus inside the fiber. This is the point at which the light has its highest intensity within the fiber.
Laser damage thresholds are lower at the surfaces than in a material’s bulk. The situation is com-
pounded if the surfaces are not properly prepared. A well polished surface will have a surface roughness
of one tenth the wavelength of the laser light. In addition, care should be taken not to embed polishing
materials in the surface. Certain techniques for cleaving, or breaking the ends of fibers produce excellent
quality faces. Under these conditions, the surface may have a damage threshold that approaches that of
the bulk. Alternatively, the fiber can be polished. Several commercial fiber polishers are available.
Connectors
Often, fibers are terminated with connectors. Connectors are fittings epoxied onto the fibers to allow
them to be quickly connected to an optical system. These precision devices allow the fiber to be removed
and replaced repeatedly without changing the alignment of the optical system. Connectors can also be
used to connect two lengths of fibers together.
Fiber Bundles
There are really two types of fiber bundle, coherent and incoherent. Coherent bundles have consistent
relative positions of all of the constituent fibers along their length, and are used in imaging endoscopes.
The resolution of the image is determined by the number of fibers in the array. Often, some of the fibers
are used for illumination and the remainder are used to carry an image back to a viewfinder or camera.
Incoherent bundles are far less expensive and are ideal for the delivery of light or laser energy. They
are sometimes used because a bundle of small fibers is much more flexible than a large fiber with the
same total core diameter. Laser power delivery bundles are used in applications such as angioplasty.
Contact Tips for Silica Fibers
Fibers are often used in conjunction with various tips that may act to shape the laser beam or cut tissue.7
Contact tips concentrate the laser energy at the distal end of the fiber and do not transmit a free beam.
The tip is placed in direct contact with the tissue, and cuts it as the laser energy is absorbed. Very little
laser energy falls on surrounding tissue. Sapphire tips can be attached to silica fibers to provide a wide
range of cutting tools. Spherical, tapered, absorbing and transmitting tips are all used for various cutting
applications with different results. Some attachable tips are actually made of silica rather than sapphire.
Some silica fibers are available with molded tips. These fibers have different shapes machined into their
distal ends as an alternative to attachable contact tips.
Launching Laser Energy into a Fiber
Typically, the laser beam is focused with one or more lenses to a spot size smaller than the fiber core
diameter. If the spatial mode of the beam is poor, it may become necessary to pass the beam through a
spatial filter, which consists of a series of lenses and apertures that expand the beam and remove its
highest order modes. After passing through this type of device, the beam can usually be focused much
more easily.
Most experts agree that the final focus of the beam should be in front of the fiber. This avoids having
the beam focus inside the fiber. If the beam were to focus in the fiber, sufficiently high intensities could
develop that would damage the fiber. At the launch face of the fiber, the beam waist should be about
70–80% of the core diameter. In addition, it is recommended that the NA of the fiber be underfilled
by 20–30%.
1.10 Optical Materials

Various materials are used to transmit light over different spectral regions. Silica fiber optics, which are
excellent for the transmission of visible light, are highly absorbing at mid-infrared wavelengths. Similarly,
materials exist that transmit infrared laser energy, but are opaque to visible light. The main factors that
determine a material’s transparency window are scattering and absorption. Absorption is the conversion
of light energy into heat. Scattering is the redistribution of light energy over different paths.
A material’s intrinsic transparency is defined by the absorption and scattering losses caused by the
molecular structure of the material. These losses are graphically represented with a V-curve. Figure 1.23
shows V-curves for oxide, chalcogenide and halide glasses. These curves were generated from data
presented by Shibata, et al.8 The minima in the V-curves are 0.16 dB/km at 1.6 µ m, 0.01 dB/km at 4.54
µm and 0.001 dB/km at 3.44 µm for oxide, chalcogenide and halide glasses, respectively.9 Due to extrinsic
factors such as bubbles, impurity absorption bands, and inclusions, the real losses in these materials may
be substantially higher, as is shown in Figure 1.24.
The intrinsic losses shown Figure 1.23 derive from three main mechanisms. The short wavelength
limit to a material’s transparency is defined by the band gap of the material. High energy photons are
101
Transmission Loss (dB/km)
multiphonon
100 edge
Oxide
10-1
electronic
transitions
Chalcogenide
10-2
Rayleigh
scatter Halide
-3
10
1 2 3 4 5
Wavelength (µm)
FIGURE 1.23 Theoretical loss curves for oxide, fluoride, and chalcogenide glasses.
10 Sapphire
Chalcogenide glass
Attenuation Coefficient (dB/m)
Hollow sapphire
waveguide
1
Silica Silver halide
Fluoride
.1 glass Laser λ (µm) α (cm-1)
HF 2.8 1000
Er:YAG 2.9 3000
CO 5.2 200
CO2 10.6 600
.01
1 2 3 4 5 6 7 8 9 10 11 12
Wavelength (µm)
FIGURE 1.24 Attenuation coefficients of various infrared transmitting fiber optics.

Basics of Lasers 21
sufficiently energetic to excite electronic processes such as valence band to conduction band transitions.
The UV-edge of the V-curve represents optical absorption due to electronic transitions.
The line in Figure 1.23 labeled multiphonon edge describes attenuation due to atomic and molecular
vibrations excited by the incident energy. The position of this line is dictated by the size of the atoms
and strength of the bonding in the material. Generally, larger atoms and weaker bonds correspond to
better IR transmission. Any light at longer wavelength than the multiphonon edge is coupled into exciting
vibrational modes, and is not transmitted through the material.
The line in Figure 1.23 labeled Rayleigh scatter describes attenuation due to microfluctuations in the
index of refraction of the material. The scattering has an A.-4 dependence that is associated with scattering
centers much smaller than the wavelength of the light. Note that the intersection of the Rayleigh scatter
and the multiphonon edge define the minimum attenuation for the optical material.
In addition to the intrinsic factors described by the v-curve, there are many extrinsic sources of optical
attenuation. Impurities may absorb or scatter light. Absorption caused by impurities might be electronic
transitions or vibrational in nature. Scattering from impurities usually arises from differences in the
indices of refraction between the impurity and the optical material. Bubbles in optical materials are said
to be optically thick because their indices are very different from those of the optical materials themselves.
Optically thick scattering centers are a strong source of scattering losses.
UV-Vis-NIR Fiber Optics
Silica-core fiber optics are used for delivery of medical laser energy in the spectral region from 200 to
2400 nm. These fibers are well developed because they have applications in telecommunications. As a
result, the optical and mechanical properties of silica fiber optics approach theoretical limits. For medical
applications, there are three varieties of silica-core fibers. While the claddings on the three varieties differ,
the cores are all SiO2, or silica.
Silica-silica, or glass-clad fiber optics, are those fibers with a silica glass cladding around a silica glass
core. The cladding glass is typically doped with fluorine or boron to lower its refractive index relative to
the core glass. Frequently, these fibers will be referred to as all-glass fibers. There are several advantages
to using silica-silica fibers rather than plastic or silicon-clad fibers.5 All-glass fibers have the lowest optical
loss coefficients of any optical fibers. In the UV and IR spectral regions, where fiber losses may be
appreciable, all-glass fibers are required for adequate transmission. These fibers also offer higher laser
damage thresholds. If the laser beam should become temporarily misaligned, or if the core is overfilled
with laser energy, a glass cladding is less likely to damage than a polymer cladding because the glass can
withstand temperatures up to 1800°C, whereas the plastic claddings can be used up to only a few hundred
degrees. This high temperature capability may also be important for hot-tip laser procedures. All-glass
fibers are also preferred for accurate beam delivery. The low NA of silica-silica fibers (NA = 0.22) provides
less beam divergence at the distal end of the fiber than high NA plastic-clad fibers (NA = 0.37). In
addition, the NAs of all-glass fibers are more stable with varying temperature than the NAs of plastic-
clad fibers because of the low expansion of silica.
For less demanding laser delivery applications, there are two less expensive types of silica-core fiber
optics. Polymer-clad silica (PCS) fibers have a soft, low index silicone cladding around a silica-glass core.
Typically a Tefzel or Nylon buffer coating is applied for extra protection. The cladding may be removed
by mechanical means, and this becomes necessary for contact tip applications. Unfortunately, the cores
of PCS fibers are free to move relative to the cladding.
This phenomenon is known as pistoning, and makes connection difficult. Hard fluoropolymer-clad silica
(TECS) fibers have a very thin, hard plastic cladding. The claddings are typically about 15 µm thick and are
protected by a buffer coating. The hard fluoropolymer actually bonds to the silica core, so no pistoning occurs.
An important parameter to consider when selecting a silica fiber for a specific application is the fiber’s
OH– content, which will affect both the spectral transmission and the cost of the fiber. Spectral trans-
mission curves of high and low OH– silica fibers are provided in Figure 1.25.5 Generally, high OH– fibers
are used for the transmission of excimer laser energy, while low OH– fibers are necessary for visible and
NIR transmission.
250
200
High OH
dB/km
150 Low OH
Transmission/Meter
97.9%
100
90.0%
50
99.8%
0
350 550 750 950 1150 1350 1550 1750 1950
Wavelength, nm
FIGURE 1.25 Effect of OH- content on silica fiber transmission.
Infrared Transmitting Fiber Optics

In the past, much of the research extolling the merits of infrared laser tissue ablation has been presented
with the disclaimer, “If only a suitable delivery system was available.” In fact, there are optical fibers available
for the delivery of energy from many of the infrared lasers that are of interest to the medical community.
While it is true that many of these fibers require more careful handling than conventional silica fiber optics,
they do offer the ability to deliver infrared laser energy for a variety of minimally invasive procedures.
Lasers such as the Er:YAG, HF, CO and CO2 operate in the infrared spectral region, where the
absorption coefficient of tissue is many times higher than at visible wavelengths. The efficiency of laser
tissue ablation is strongly dependent on the absorption coefficient of the target tissue. In addition, strongly
absorbed laser energy creates less thermal damage in surrounding tissue. Therefore, infrared laser surgery
represents a relatively precise method of tissue removal. The development of infrared fiberoptic catheter
technology may result in a multitude of new, noninvasive therapeutic procedures.
Figure 1.24 shows the attenuation spectra of some of the fibers currently available for delivery of
infrared laser energy. Also shown in this figure are the emission wavelengths of several clinically useful
lasers and the approximate absorption coefficients of tissue at these wavelengths.10 The attenuation spectra
shown account only for the propagation loss in the fibers, reported in units of attenuation per length.
Fresnel losses caused by reflections from the launch and delivery faces of the fiber are neglected. Generally,
an attenuation coefficient of 1 dB/m or less is considered acceptable for delivery of energy over a distance
of several meters. The following sections describe each of the types of fiber depicted in Figure 1.24.
Table 1.7 summarizes the properties of the fibers.
Fluoride Glass Fiber Optics
Heavy-metal fluoride glass (HMFG) optical fibers are similar to conventional silica fiber optics in many
ways. Like conventional fibers, they possess a glass-core/glass-cladding structure, have very low optical
losses and are quite flexible. In addition, fluoride glasses offer transparency through the visible portion
of the spectrum out to about 4.5 µ m wavelength.11 These fibers have a demonstrated ability to deliver
high energy pulses from the Er:YAG laser. Fibers with core diameters of 250 µ m have been used to deliver
450 mJ pulses (900 J/cm2) of laser energy at 2.94 µ m wavelength for short periods of time.12–14
Unfortunately, the extended infrared transparency offered by fluoride glasses is derived from the
material’s relatively weak molecular bonding.15 Thus, the same glass structure that is blessed with a wide
transparency window is, at the same time, cursed with a low maximum use temperature (150° C), limited
mechanical strength and some degree of chemical reactivity.
The past 10 years have seen an enormous push to further develop fluoride glasses. Much of this research
was fueled by the extremely low intrinsic optical attenuation coefficients of the material, and directed at
fulfilling the objective of a repeaterless transoceanic fiberoptic telecommunications link. While this goal
has yet to be realized, many advances have been made in the field. Fluoride fibers with minimum
Basics of Lasers 23
attenuation coefficients of several dB/km are readily available. For medical applications involving only a
few meters of material, the fibers are essentially lossless.
One of the major frustrations in working with fluoride glasses has been the chemical reactivity of the
fibers. The industry standard fluorozirconate, or ZBLAN, fibers are somewhat soluble in water and so
degrade rapidly in an aqueous environment. In addition, laser damage to the fiber end-faces during the
delivery of high fluences of Er:YAG laser energy has been associated with a hydrolysis reaction. Compo-
sitions of fluoride glass with improved resistance to attack by water are traditionally more difficult to
manufacture in fiber form.16,17 Recently, however, low-loss fibers with enhanced chemical durability have
been produced.18 Fibers fabricated from these new compositions have substantially higher laser damage
thresholds for the Er:YAG laser14 and are available from at least one commercial source.
Single-Crystal Sapphire Fiber Optics

Single crystals of Al203, or sapphire, which are grown into optical fibers, offer many advantages for the
delivery of Er:YAG laser energy. Sapphire is extremely hard, has a melting temperature greater than
2000°C, is biologically inert and does not degrade during laser delivery. The fibers can be used in direct
contact with tissue.
Unlike silica and fluoride glasses, however, sapphire fibers are grown without an optical cladding.
Core-only fibers are susceptible to excessive loss of laser energy to the fiber environs, unless they are
properly jacketed. Teflon and other polymers have been applied to sapphire fibers for this purpose,19 and
further research on optical cladding materials is ongoing.
Sapphire can be grown into single-crystal fibers by one of two methods. The edge-defined, film-fed
growth process (EFG) has been used to grow sapphire fibers for structural reinforcement applications
for nearly 20 years.20 Unfortunately, structural sapphire fiber typically has high optical losses and is
unsuitable for laser power delivery. Efforts to produce optical quality EFG sapphire fiber are currently
under way, and attenuation coefficients of less than 2 dB/m have recently been measured with the Er:YAG
laser [J. Fitzgibbon, Saphikon, Inc., Milford, NH. Private communication 1992]. EFG sapphire fiber is
offered commercially, but optical quality fiber is not yet available.
Laser-heated pedestal growth (LHPG) of single-crystal sapphire has been shown to be a viable process
for producing optical quality fibers.19,21 Fibers with optical attenuation coefficients of less than 1 dB/m
at the Er:YAG laser wavelength are readily produced. LHPG sapphire fibers with core diameters of 340
µm have been used to deliver 600 mJ pulses of energy from the Er:YAG laser.19 Unfortunately, there are,
as yet, no commercial sources for LHPG sapphire.
Chalcogenide Glass Fiber Optics

Chalcogenide glasses contain the group VI elements S, Se or Te as the network-forming anion.22 These
glasses are generally opaque to visible radiation, but transmit well in the near- and mid-infrared spectral
regions. Optical fibers fabricated from chalcogenide glass compositions have been used to deliver several
hundred watts of CO laser radiation at wavelengths between 5.2 and 5.9 µ m.23 The fibers can also be
used to transmit radiation from the CO2 laser, but laser damage from a phenomenon known as thermal
lensing limits the deliverable power to about 10 W.
Traditionally, chalcogenide glass fibers have been extremely brittle and difficult to work with. However,
recent advances in cladding and coating technology have revolutionized this field.24 New chalcogenide
fibers are available that offer good flexibility and strength. Like all chalcogenide glasses, the new fibers
have a low use temperature and should not be exposed to temperatures much above 100°C.
Chalcogenide glasses have good chemical durability at room temperature and are not subject to attack
by water. Chalcogenide glass fibers are available commercially from a variety of sources. Unfortunately,
the properties of fibers from different vendors vary substantially. A range of chalcogenide fiber properties
is included in Table 1.1.
Polycrystalline Fiber Optics

Polycrystalline (PC) fiber optics have been fabricated from the halides of silver, thallium and potassium.
The materials are extruded through polished dies at elevated temperature and pressure. Silver halides
(AgCl-AgBr) are currently the best PC fibers for the delivery of CO and CO2 laser energy. Clad and
unclad silver halide fibers are readily produced with attenuation coefficients of 3 and <1 dB/m at 10.6
µm wavelength, respectively.25,26 Clad fibers deliver energy with better beam quality and are less sensitive
to hostile delivery environments. Unfortunately, irregularities at the core-cladding interface cause optical
scattering and increase the total attenuation in these fibers. Severe aging problems have also been
associated with polycrystalline fibers. The optical properties of the fibers degrade over time.
Packaging of silver halide fibers is critical for several reasons. The fibers are corrosive to many materials,
and are quite sensitive to ultraviolet radiation. Exposure to fluorescent room light causes a dramatic
increase in the attenuation coefficients of the fibers. In addition, the fibers will permanently deform if
bent beyond a certain point. Fortunately, delivery catheters have been developed to protect the fibers
from such damage. Most often, the fibers are found in hand pieces at the end of articulated arms used
with surgical CO2 lasers. Laser damage constraints generally limit these fibers to delivering about 10 W
of CO2 laser energy.26
Hollow Waveguides
Hollow waveguides fall into two general categories, leaky and guided-mode propagating waveguides.27
Both types transmit laser energy through the hollow bore of a tubular fiber. Leaky waveguides are made
from metals or dielectric coated metals, and transmit energy along a highly reflective inner surface. In
some cases, both metallic and dielectric coatings are applied to a substrate to create a leaky guide.28
Guided-mode hollow waveguides operate on the principle of total internal reflection, much like conven-
tional fiber optics. Sapphire,29 polycrystalline alumina,30,31 and several oxide glasses32 have been used as
guided-mode waveguides. Both guided-mode and leaky waveguides are effective for delivery of CO2 laser
power. Because the laser power propagates in air rather than in a solid, laser damage thresholds are high,
and Fresnel reflection losses are nonexistent.
Guided-mode waveguides can be thought of as optical fibers with air as the core. At certain wavelengths,
the refractive index of specific materials may dip below unity because of a phenomenon known as
anomalous dispersion. At these wavelengths, tubes of the material can act as waveguides with an air core
(nair = 1). Sapphire tubes have been used as waveguides for CO2 lasers with attenuation coefficients of
less than 0.5 dB/m.29 Interestingly, sapphire hollow waveguides are single-mode waveguides, which allows
their output energy to be refocused easily. Doped silica tubes have also been used as guided-mode hollow
waveguides, but their attenuation coefficients have been high.33,34
Dielectric coated metal leaky guides have demonstrated the ability to deliver CO2 laser radiation
with attenuation coefficients of less than 0.1 dB/m.35 These waveguides have been fabricated with both
circular and rectangular cross sections. A water-cooled version has been used to deliver almost 3000
W of power from a CO2 laser.36 Multilayer coated plastic guides are susceptible to laser damage at
powers greater than 50 W.
Hollow waveguides generally have very limited flexibility. Most often, they are used in hand pieces and
connected to articulated arms from surgical CO2 lasers. One commercial hollow waveguide boasts a 50 cm
minimum bend radius. Some fixed-curve sections of hollow waveguides are available for specific applications.
1.11 The Future of Medical Lasers and Fiber Optics

As accurate as the current coverage is in describing the current state of products commonly available for
medical applications, a new wave of laser and fiber optic devices is beginning to make its way from the
research labs. This new technology will soon begin to replace everything currently described. In a few
years, medicine and biology will be flooded with new technology, briefly outlined in the next several pages.
In a nutshell, here is what will happen. Solid state devices will replace all gas lasers wherever possible.
Semiconductor diodes will either be used directly for applications within their power range or will be used
as pumping sources for solid state laser materials. Solid state diodes are the material of choice because of
their efficient conversion from electricity to light. New medical devices will consist of the following:
Basics of Lasers 25
Femtosecond lasers — Ti:sapphire as well as other materials will enable exceedingly high power lasers,
low energy pulsed systems that will allow surgeons to perform precise surgery without heating tissue as
much. The high powers will allow table top accelerators that will generate x-rays, protons, and new
nuclear isotopes, and enable technologies for medical diagnostics.
New fiber lasers — Fiber lasers can also take advantage of solid state diode pumping. Fiber lasers, or
lasers in which the fiber core becomes the lasing material and the accompanying fiber-cladding becomes
the element that contains losses and guides the laser build-up without the need for a conventional resonant
cavity, makes simple devices possible. Smaller, less expensive lasers and higher power from a reduced
footprint system will make it easier for the laser to get into the operating room.
All solid state, complete coverage of the visible and near infrared region — Solid state pumped solid
or fiber lasers will have greater power and will cover all wavelengths. The high peak power from femto-
second laser will allow more use of non-linear optics and the generation of nearly every wavelength
needed in the visible and infrared for those surgical and photochemical (photodynamic and therapeutic)
applications.
Mid-infrared semiconductor lasers — These devices are also emerging from the research lab and will,
if cooling problems can be solved, make excellent devices for gas and thin tissue sample analysis. The
mid-infrared has the capability of determining exact chemical specificity and could possibly enable optical
disease characterization.
In other words, the next 10 years will see nearly all solid state lasers giving higher peak powers; higher
average powers; more efficient operation covering the visible, the near IR, the ultraviolet and large parts
of the vacuum ultraviolet, x-ray and the mid- and far-infrared as well. Most of the gas lasers will be
replaced, as will dye lasers, chemical lasers and other less convenient lasers.
Fiber optics could also take a great leap forward, if claims have merit. Photonic crystal type hollow
waveguides are being touted in the popular press as having made a leap to low transmission loss
approaching 0.01 db loss per kilometer. This would perhaps greatly improve both communications and
medical applications, but one must be cautious until the results appear in professional journals and have
been proven true. If these predictions hold true, the medical field will likely soon have vastly new types
of sources and delivery systems.
References
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9. Tran, D.C., Sigel, G. H., Jr. and Bendow, B., Heavy metal fluoride glasses and fibers: a review, J.
Lightwave Technol., LT-2 (5) pp. 566–586, 1984.
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11. Drexhage, M.G., Infrared glass fibers, Proc. S.P.I.E. 1228, pp. 2–11, 1990.
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S.P.I.E. 1048, pp. 141–144, 1989.
13. Kwark, B. et al., An analysis of Er:YAG laser for angioplasty: delivery system, ablation capability,
and the effect of water content, Lasers in the Life Sciences 5, pp. 113–128, 1992.
14. Merberg, G.N. et al., Evaluation of crystalline and chemically durable glass fibers for Erbium-YAG
laser delivery systems, Proc. S.P.I.E. 1228, pp. 216–223, 1990.
15. Drexhage, M.G. and Moynihan, C.T., Infrared optical fibers, Scientific American 259, pp. 110–115,
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16. Kanamori, T. et al., BaF2-CaF2-YF3-AlF3 based glass systems for infrared transmission, Japan. J.A.P.
20, pp. 326–328, 1981.
17. Shahriari, M.R. et al., Fabrication of AlF3-based glass fibers, Proc. Opt. Fiber Mat. and Processing
Symp. 172, pp. 163–168, 1990.
18. Iqbal, T. et al., AlF3-based glass fibers with enhanced optical transmission, Electronics Lett. 27, pp.
2-3, 1991.
19. Merberg, G.N. and Harrington, J.A., Optical and mechanical properties of single-crystal sapphire
optical fibers, Appl. Opt. 32, pp. 3201–3209, 1993.
20. LaBelle, H.E., Jr., EFG, the invention and application to sapphire growth, J. Crystal Growth 50, pp.
8–17, 1980.
21. Jundt, D.H., Fejer, M.M. and Byer, R. L, Characterization of single-crystal sapphire fibers for optical
power delivery systems, Appl Phys. Lett. 55, pp. 2170–2172, 1989.
22. Nishii, J. et al., Recent advances and trends in chalcogenide glass fiber technology: a review, J. Non-
Crystalline Solids 140 199–208,1992.
23. Sato, S. et al., Multihundred watt CO laser power delivery through chalcogenide glass fibers, Appl.
Phys. Lett. 62 669-671, 1993.
24. Hilton, A.R., Sr., Chalcogenide glass optical fibers, Proc. Soc.Photo-Optical Instr. Engrg. 1591, 32–42,
1992.
25. Artjushenko, V. et al., Infrared cables and catheters for medical applications, Proc. Soc. Photo-optical
Instr. Engrg. 1420, 157–168, 1991.
26. Paisss, I., Moser, F. and Katzir, A., Core-clad silver halide fibers for CO2 laser power delivery, Proc.
Soc. Photo-Optical Instr. Engrg., 1420, 141–148, 1991.
27. Miyagi, M., Consideration on realization of low-loss total reflection-type hollow-core fiber at mid-
infrared, Proc. Soc. Photo-Optical Instr. Engrg. 843, 76–79, 1987.
28. Dror, J., et al., Hollow plastic waveguides internally coated with metal and dielectric films, Proc.
Soc. Photo-Optical Instr. Engrg. 843, 88-93, 1987.
29. Harrington, J.A. and Gregory, C.C., Hollow sapphire fibers for delivery of CO2 laser energy, Optics
Lett. 15, 541–543, 1990.
30. Harrington, J.A., Gregory, C.C. and Nubling, R., Hollow waveguides for CO2 laser delivery systems,
Proc. Soc. Photo-Optical Instr. Engrg., 1048, 117–121, 1989.
31. Gregory, C.C. et al., Hollow curved Al2O3 waveguides for CO2 laser surgery, Proc. Soc. Photo-Optical
Instrumentation Engrg. 1420, 169–175, 1991.
32. Worrell, C.A., Infrared optical properties of glasses and ceramics for hollow waveguides operating
at 10.6 µm wavelength, Proc. Soc. Photo-Optical Instr. Engrg. 843, 80–87, 1987.
33. Harrington, J.A. and Katzir, A., Specialty fibers tackle tough problems, Laser Focus World 6, 139,
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34. Saggese, S.J., Harrington, J.A. and Sigel, G.H., Jr., Attenuation of incoherent infrared radiation in
hollow sapphire and silica waveguides, Optics Lett. 16, 27–29, 1991.
35. Miyagi, M., Hongom A., and Matsuura, Y., Hollow IR waveguides, Proc. Soc. Photo-Optical Instr.
Engrg. 1228, 26–35, 1990.
36. Hongo, A. et al., Transmission of kilowatt-class CO2 laser light through dielectric-coated metallic
hollow waveguides for material processing, Appl. Optics 5114–5120, 1992.
2
Optical and Thermal
Response of Tissue
to Laser Radiation
2.1 Introduction ......................................................................... 27

2.2 The Optical Response of Tissue.......................................... 28
A.J. Welch Scattered Light • Reflection • Reflected Power • Semi-Infinite
Slab • Low Albedo • High Albedo • Rate of Heat Generation •
University of Texas
Selection of Wavelength • Time Resolved Propagation
Craig Gardner 2.3 Thermal Response................................................................ 41
Rio Grand Medical Error Analysis
Technologies, Inc. 2.4 Summary............................................................................... 45
References ........................................................................................ 45
2.1 Introduction
A unified theory for the interaction of laser light with tissue is not possible at this time. However, it is
possible to describe some basic interactions that provide some small insight into the effect of laser light
on tissue. When an overall problem overwhelms analysis, engineers are taught to subdivide it into
components that can be analyzed individually.
Typically, laser–tissue interactions have been divided into the optical response and the resulting thermal
response. Actually, considerable progress has been made on many basic aspects of laser–tissue interaction,
and a few real problems have been analyzed. However, it has been difficult to string basic concepts together
to describe nonlinear interactions such as tissue ablation, or situations where the physical properties of
tissue do not remain constant during laser radiation.
In this chapter, we will examine basic optical and thermal interactions. The applications and limitations
of this analysis will be explored in terms of published experimental data. Typically, absorbed light is
converted to heat; however, light absorbed by fluorophores or photosensitizers can produce fluorescence
or photochemical reactions, respectively.
A wide range of tissue reactions can be achieved by varying the laser pulse duration, energy per pulse,
wavelength and spot size. In each case, the amount of energy absorbed in a specified volume is critical.
The rate of heat generation S(r) at a position r(r = x,y,z) is equal to
S(r) = µa(r)φ (r) (2.1)
where φ (r) is the local fluence rate [W/m2] and µa is the local absorption coefficient [1/m] or [m–1]. The
total fluence rate includes both the fluence rate of scattered light and the fluence rate of the attenuated
collimated laser beam at r.
0-8493-1146-2/02/$0.00+$1.50
2.2 The Optical Response of Tissue

If tissue is irradiated with a laser beam, the fluence rate [W/m2] of the beam inside the tissue decreases
exponentially (Beer’s law). The attenuation of the laser beam is due to absorption and possibly scattering
of the collimated light according to the relation
–µt z
E ( z ) = ( 1 – r s )E 0 e (2.2)
where
E(z) is the attenuated beam within the tissue[W/m]
E0 is the irradiance [W/m2]
µt is the attenuation coefficient [1/m]
rs is the Fresnel specular reflection for non-polarized light which is about 2% for light normal to
an air–tissue interface.
Laser–tissue interaction is viewed as the propagation of photons through a volume of randomly

distributed absorption and scattering centers. In the framework of this representation, the probability
that a photon will be absorbed or scattered over a distance ∆S is given by µt∆S. Further, the probability
that the photon is absorbed over the distance ∆S is µa∆S, where µa is the absorption coefficient with
units [1/m]. Likewise, it follows that the probability of a scattering event over ∆S is µs ∆S, where µs is the
scattering coefficient with units [1/m]. Based on these definitions and assuming that absorption and
scattering are disjoint events
µt = µa + µs (2.3)
Although Equation (2.2) assumes a continuous laser beam, Beer’s law holds for pulsed irradiation.
–µt z
H ( z ) = ( 1 – r s )H 0 e (2.4)
where H0 is the radiant exposure [J/m2] and H(z) is the energy per unit area of the attenuated laser beam
[J/m2] at a distance z within the tissue.
2.2.1 Scattered Light

The radiance of scattered light, L(r, s), [W/(m2 ⋅ sr)] at r in unit direction s is reduced by absorption and
by scattering of light from direction s to other directions s′ as the light travels a distance ds. However,
light is gained by scattering from other directions s' into direction s. The scattering from s′ to s is described
by the single scattering phase function p(s, s′ ), which has units [1/sr].
For homogeneous tissue, we assume that the phase function can be described by the angle θ between
s′ and s and does not depend on the initial direction s′ . That is,
p(s, s′) = p(s ⋅ s′) = p(cosθ ) (2.5)
The expected (probability weighted) cosine of the scattering angle θ is given by
∫
( cos θ )p ( cos θ ) d( cos θ )
g = ------------------------------------------------------------- (2.6)
∫
p ( cos θ ) d( cos θ )
Light in tissue is forward scattered, and typically the value of g is between 0.85 and 0.99.
Optical and Thermal Response of Tissue to Laser Radiation 29
In concept, light propagation in a scattering medium can be described by the transport equation that
describes the transfer of energy1
dL ( r, s )
∫
------------------- = – µ t L ( r, s ) + µ s p ( s, s′′ )L ( r, s′′ ) dω′ [ W ⁄ m ⋅ sr ]
3
(2.7)
ds
Solutions of the transport equation include the exact adding-doubling method for one-dimensional
problems,2 or the not-so-exact diffusion approximation for three-dimensional problems.1,3 However,
most attempts to model light propagation in tissue rely on Monte Carlo simulations.4,5
The fluence rate of scattered light at r is obtained by summing all of the light (radiance) through the
point r. That is,
∫ L ( r, s ) dω [ W ⁄ m ]
2
φs ( r ) = (2.8)
4π
The importance of the contribution of scattered light to the spatial distribution of the rate of heat
generation depends not only on the absorption coefficient at location of r but the relative ratio of the
scattering coefficient to the attenuation coefficient. This ratio is called the albedo, a, where
µ µs
a = ----s = ---------------
- (2.9)
µt µa + µs
If a is greater than about 0.8, then the diffusion approximation provides a reasonable estimate of the
distribution of scattered photons. When the albedo is less than 0.6, photons scattered from the collimated
beam tend to be absorbed within or near the beam path, especially for beam diameters larger than the
penetration depth of the beam, δ, where
1
δ = ---- [m] (2.10)
µt
2.2.2 Reflection
The reflection of light at an interface can be described by considering a ray of light at angle θ1, with
respect to the surface normal as depicted in Figure 2.1. When the index of refractions of the two media
are not matched, then the angle of reflection is θ1 and the angle of transmitted light θ2 is given by Snell’s law
n 1 sin θ 1 = n 2 sin θ 2 (2.11)
specular reflection
θ1 θ1
air, n1
tissue, n2
θ
2
transmitted into tissue
FIGURE 2.1 Reflectance and transmittance of a nonpolarized ray of light incident upon an interface with index of
refraction n1 for the top layer and n2 for the bottom layer. With permission from Kluwer Academic.17
At a boundary, the reflection of a ray of non-polarized light at angle θ1 with respect to the surface normal
is given by the Fresnel law as
2 2
1 sin ( θ 1 – θ 2 ) tan ( θ 1 – θ 2 )
r f ( θ ) = --- ------------------------------
- + ------------------------------- (2.12)
2 sin 2( θ 1 + θ 2 ) tan 2( θ 1 + θ 2 )
2.2.3 Reflected Power

The incremental power dP(r, s) flowing in direction s with infinitesimal solid angle dω through an
infinitesimal area dA located at r is
dP ( r, s ) = L ( r, s ) cos θdAdω (2.13)
where θ is the angle between s and a normal to the surface of dA. If the diffuse light is isotropic, then
L ( r, s ) = L 0 (2.14)
The reflected power per unit area for diffuse (isotropic) light incident between θ1 and θ1 + dθ1 on the
interface of two layers is
2πr f ( θ 1 )L 0 cos θ 1 sin θ 1 dθ 1 (2.15)
where rf(θ1) is the Fresnel reflection coefficient at θ1. The specular reflection of diffuse light incident on
an air–tissue interface is given by
power of reflected light

r sd = -------------------------------------------------------- (2.16)
power of incident light
For irradiance with diffuse light
π⁄2 π⁄2
2π ∫ r f ( θ 1 )L 0 cos θ 1 sin θ 1 dθ 1 πL 0 ∫ r ( θ )2 sin θ cos θ dθ

f 1 1 1 1
0 0
r sd = ---------------------------------------------------------------------
- = ----------------------------------------------------------------------
π⁄2
πL 0
∫
2π L 0 cos θ 1 sin θ 1 dθ 1 (2.17)
0
π⁄2
= ∫ r ( θ ) sin ( 2θ ) dθ
f 1 1 1
0
When light travels from layer 2 (tissue) to layer 1 (air), all rays at an angle θ2 greater than a critical
angle θc are totally reflected. That is,
r f ( θ 2 ) = 1 for θ 2 ≥ θ c (2.18)
The fraction of light internally reflected, rid, is given also by Equation (2.16). Unknown is the form
of the radiance at the interface owing to the backscattering of light in tissue. If we assume that it is
isotropic, then radiance can be represented by a constant. Thus the equation for internal reflection,
rid is
θc π⁄2
∫
2π r f ( θ 2 )L 0 cos θ 2 sin θ 2 dθ 2 + 2π ∫ L cos θ sin θ dθ
0 2 2 2
0 θc
r id = ---------------------------------------------------------------------------------------------------------------------------------
πL 0 (2.19)
θc
1
= ∫ r ( θ ) sin ( 2θ ) dθ
f 2 2 2 + --- [ cos ( 2θ c ) + 1 ]
2
0
The critical angle θc is given by Snell’s law for θ1 = 90° as
n
θ c = arc sin  -----1 (2.20)
 n 2
The critical angle for total internal reflection in tissue at an air boundary is about 45° for ntissue = 1.4.
*
An approximation r id for r id can be obtained by assuming rf(θ2) = 0 for θ2 < θc. Using Equation (2.19),
* 1
r id = --- [ cos ( 2θ c ) + 1 ]
2
(2.21)
2 1
= cos θc = 1 – -----2
n
*
where n = n2/n1. r id is a lower bound on the fraction of diffuse (isotropic) light that is internally reflected
at the boundary.
The importance of index refraction upon reflectance is illustrated in Figure 2.2 for three situations:
(1) reflectance of collimated light normal to the interface; (2) diffuse light incident upon tissue rsd; (3)
internal reflection of diffuse light within tissue rid.
When the media have similar boundaries, such as a water–tissue interface, and internal reflection
is not significant, the backscattered light leaves the tissue. This can produce a subsurface peak in
fluence rate just below the surface when the albedo is large and the spot size is much larger than
the penetration depth.
2.2.4 Semi-Infinite Slab

Many of the features of light propagation in tissue can be illustrated by considering a slab geometry
where the laser beam is normally incident on the surface of the slab. Although the penetration of the
light in the slab is primarily determined by the optical properties of the slab, the radius of the beam
influences penetration depth. These effects are illustrated in a series of figures where Monte Carlo
simulations have been used to estimate the total light distributions (collimated plus scattered light) in
an infinite slab with an air–tissue interface at the surface of the slab.
2.2.5 Low Albedo

An interesting and perhaps misunderstood situation occurs when the albedo is low, say 0.3 ≤ a ≤ 0.6. In
this range, neither absorption nor scattering dominates. Light scattered from the laser beam is usually
absorbed in the vicinity of the beam. Assuming a uniform irradiation of the slab simplifies light propa-
gation to a one-dimensional problem, and it is reasonable to expect the light to approximately decrease
exponentially with depth. That is,
–k2 z
φ ( z ) = k1 e (2.22)
Tissue Reflection Coefficient

Collimated and Diffuse Light
r id
0.8
surface collimated, rsc
surface diffuse, rsd
0.7 internal (approx)
internal (integral),rid
r id
0.6 (approximation)
0.5
Reflectance
0.4
0.3
0.2 r sd
0.1
r sc
0.0
1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0
Tissue Index of Refraction (relative to air interface)
FIGURE 2.2 Boundary reflectance for (1) collimated light normal to surface of boundary, (2) diffuse light incident
upon surface from region of low index of refraction, rsd, and (3) internal reflection of isotropic diffuse light from
region of high index of refraction, rid. The rid approximation is based on Equation (2.21). With permission from
Kluwer Academic.17
Certainly k1 is not going to be much larger than E0(1 – rs) where E0 is the irradiance and rs is the specular
reflectance at the surface.
Suppose rs = 0 and k1 = E0 and the total fluence follows Equation (2.22). The value of k2 follows from
a simple energy balance. The total power absorbed per unit area is
∞ ∞
–k2 z µa E0
∫ S ( z ) dz = ∫ µ E e
a 0 dz = ----------
k2
- = E0 (2.23)
0 0
For the assumed conditions, k2 must equal µa. Thus, the distribution of total fluence rate (collimated
plus scattered light) is
–µa z
φ ( z ) = E0 e (2.24)
Equation (2.24) may be somewhat unexpected since the collimated light decreases according to Equation
(2.1). For this one-dimensional example:
– ( µ a + µ s )z
E ( z ) = E0 e (2.25)
When the albedo is between 0.3 and 0.6, k1 is slightly larger (about 5%) than E0, the attenuation is
not exactly exponential, and any exponential curve fit has a coefficient k2 slightly larger (also about 5%)
than µa . For example, see Figure 2.3 for a = 0.5, g = 0.9 and an infinite beam radius. The
–µ z
( 1 – r 2 )E 0 e a curve of Figure 2.3 is not a bad approximation for the one-dimensional case, and satisfies
the energy balance requirement.
a
1
r = 0.01 cm
r = 0.03 cm
0.8 r = 0.1 cm
r=
8
Fluence rate on axis,
φ(z) [W cm-2]
0.6
(1 -rs)exp(-µaz)
0.4 (1 -rs)exp(-µtz)
0.2 a = 0.5
µa = 10 cm-1
g = 0.9
0
0 0.05 0.1 0.15 0.2
Depth, z [cm]
b
1
r = 0.01 cm
r = 0.03 cm
0.8 r = 0.1 cm
r =
8
φ(z) [W cm-2]
0.6
(1 -rs)exp(-µaz)
0.4 (1 -rs)exp(-µtz)
0.2 a = 0.33
µa = 10 cm-1
g = 0.9
0
0 0.05 0.1 0.15 0.2
Depth, z [cm]
FIGURE 2.3 Monte Carlo simulation of total fluence rate at r = 0 as a function of depth for various beam sizes in
a semi-infinite medium with air–tissue interface. Computations were made for uniformly distributed collimated laser
–µ z –µ z
beam. Tissue optical properties were µa = 10 cm–1 and g = 0.9. Curves of ( 1 – r s )E 0 e a and ( 1 – r s )E 0 e t provide
bounds for the simulated fluence rates. Computed for albedos of (a) 0.5, (b) 0.33 and (c) 0.6. (continued)
Equation (2.22) is still a reasonable approximation for total fluence rate along the center line of the
beam path in tissue, even when the spot size is small. Note that the total fluence, φ, is approximately
–µa z – ( µ a + µ s )z
bounded from above by e and below by e as beam radius goes from ∞ to zero. The influence
of albedo is seen in Figure 2.3b for a = 0.33 and in Figure 2.3c for a = 0.6. In all Monte Carlo simulations,
µa = 10 cm–1 and g = 0.9.
2.2.6 High Albedo

The chromophores that primarily affect wavelength-dependent absorption of light in the visible and IR
spectrum of tissue are melanin, blood and water. However, between 600 nm and 1.2 µm, the absorption
coefficient of these chromophores is usually much, much less than the scattering coefficient of a tissue.
Values for the albedo are typically from 0.75 to 0.99. Although a collimated beam is attenuated in a few
c
1 r = 0.01 cm
r = 0.03 cm
r = 0.1 cm
0.8 r =
8
φ(z) [W cm-2]
0.6
(1 -rs)exp(-µaz)
(1 -rs)exp(-µtz)
0.4
0.2 a = 0.6
µa = 10 cm-1
g = 0.9
0
0 0.05 0.1 0.15 0.2
Depth, z [cm]
FIGURE 2.3 (CONTINUED) Monte Carlo simulation of total fluence rate at r = 0 as a function of depth for various
beam sizes in a semi-infinite medium with air–tissue interface. Computations were made for uniformly distributed
–µ z
collimated laser beam. Tissue optical properties were µa = 10 cm–1 and g = 0.9. Curves of ( 1 – r s )E 0 e a and
–µt z
( 1 – r s )E 0 e provide bounds for the simulated fluence rates. Computed for albedos of (a) 0.5, (b) 0.33 and (c) 0.6.
optical depths, the resulting scattered light may propagate through several centimeters of tissue. Away
from the collimated beam, the diffusion approximation can be used to describe the light distribution.
The time varying diffusion approximation is
1 ∂φ ( r, t ) 2
--- ------------------ = D∇ φ ( r, t ) – µ a ( r )φ ( r, t ) + S 0 ( r, t ) (2.26)
c ∂t
where c is the speed of light in tissue, S0 is the source term and D is the diffusion constant.
1
D = ----------------------------------------- (2.27)
3 [ µa + µs ( 1 – g ) ]
For steady state optics, ( ∂φ ) ⁄ ( ∂t ) = 0 . A complete discussion of the diffusion approximation can be
found in Refs. 1,3.
The one-dimensional steady state solution (uniform irradiance E0 over the slab) for scattered fluence
rate φs(z) for a semi-infinite slab has the form
φs ( z ) –µ z –µ z
------------ = Ae eff + Be t (2.28)
E0
where A and B are constants that depend on the boundary conditions and the effective attenuation
coefficient, µeff,
µ eff = 3µ a [ µ a + µ s ( 1 – g ) ] (2.29)
The total fluence rate φ(z) that includes the collimated light is
φ(z) φ –µ z
---------- = -----s ( z ) + e t (2.30)
E0 E0
4
φ(z)/Eo = φs(z)/Eo + exp(-µtz)
Bexp (-µeffz)
3 φs/Eo

2
φ(z)/E [-]
1
O
exp(-µtz)
0
-1
-2
Aexp(-µtz)
-3
0 0.005 0.01 0.015 0.02 0.025 0.03
Depth, z [cm]
FIGURE 2.4 Form of one-dimension diffusion approximation solution for the fluence rate of the scattered light,
–µ z –µ z –µ z
φ s ( z ) = Ae t + Be eff , and attenuation of the collimated beam, e t . Solution is for collimated irradiance of a
semi-infinite medium with an air–tissue interface.
When the albedo is large,
µ eff < µ t (2.31a)
and, for many boundary conditions including index matched boundaries,
A>0 (2.31b)
B<0 (2.31c)
A+B>0 (2.31d)
– µ eff z
As z becomes large, the total fluence rate is approximated by Ae . In fact, the basic definition
of µeff is not Equation (2.29), but rather the actual exponential attenuation coefficient of diffuse light
with depth.
The one-dimensional solution for the conditions of Equation (2.30) has the form depicted in Figure 2.4
for E0 = 1 and rs = 0.025. The curves were determined for the conditions of Figure 2.5; mismatched
boundary µa = 10 cm–1, a = 0.95 and g = 0.9.
– µ eff z
As expected, the fluence rate is attenuated by Ae as z becomes large. Perhaps not expected is (1)
the subsurface peak and (2) the fluence rate below the surface are larger than the input irradiance. In
the limit as µa → 0, the fluence rate just below the surface φ(0+) → 3 for matched boundary conditions.3
2.2.7 Rate of Heat Generation

Coagulation and ablation are governed by the rate of heat generation, S(r). As stated in Equation (2.4),
the rate of heat generation is proportional to the product of the local absorption coefficient and local
fluence rate. An interesting question is the effect of scattering on S(r). In the following set of Monte
Carlo simulations, the surface irradiance is 1 W/cm2, the absorption coefficient of the semi-infinite,
thick slab is 10 cm–1, and the anisotropic factor, g, is 0.9. There is an index of refraction mismatch at
the top surface to simulate an air (nair = 1)–tissue (ntissue = 1.38) interface. Parameters are beam radius
(0.01, 0.05 and 2.0 cm) and albedo (0.5, 0.8 and 0.95). The irradiance is assumed to be uniform within
the beam radius.
a = 0.5
r = 0.2 cm
0
10
0.05
0.1
Depth [cm]
0.15
1.0
0.2
a
0.1
0.25
0.3
0.01
0.35
0.4
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Radius [cm]
a = 0.85
r = 0.2 cm
0
10
0.05
0.1
1.0
Depth [cm]
0.15
0.2 b
0.1
0.25
0.3
0.01
0.35
0.4
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Radius [cm]
a = 0.95
r = 0.2 cm
0
10
0.05
0.1
1.0
Depth [cm]
0.15
0.1
0.2
0.01
c
0.25
0.3 0.001
0.35
0.4
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Radius [cm]
FIGURE 2.5 Monte Carlo simulation of the rate of heat generation for a beam radius of 0.2 cm. Irradiation
parameters: Uniform irradiance E0 = 1 W/cm2, µa = 10 cm–1, g = 0.9 and mismatched boundary (n2/n1 = 1.38). The
rate of heat generation is computed for albedos of 0.5, 0.85 and 0.95.
In the absence of scattering, the rate of heat generation just below the surface is 10 W/cm3
(neglecting specular reflection for collimated light normal to the surface). The rate of heat generation
decreases according to Beer’s law and the 1/e penetration depth is 0.1 cm. S(z) decreases to 1 W/cm3
at a depth of 0.23 cm. When scattering is zero, these values hold for any spot size as long as the
irradiance is 1.0 W/cm2.
a = 0.5
r = 0.05 cm
0
10
0.05
1.0
0.1
0.1
Depth [cm]
0.15
0.2
a
0.01
0.25
0.3 0.001
0.35
0.4
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Radius [cm]
a = 0.8
r = 0.05 cm
0
10
0.05
1.0
0.1
0.1
Depth [cm]
0.15
0.2 0.01 b
0.25
0.001
0.3
0.35
0.4
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Radius [cm]
a = 0.95
r = 0.05 cm
0
10
0.05
1.0
0.1
0.1
Depth [cm]
0.15
0.01
0.2
0.001 c
0.25
0.3
0.35
0.4
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Radius [cm]
For the Monte Carlo results presented in Figures 2.5, 2.6, and 2.7, the albedo is varied by changing
the scattering coefficient and holding the absorption coefficient constant; the beam radius is 0.2, 0.05
and 0.01 cm respectively in these figures. For a large spot size (Figure 2.5), the rate of heat generation at
the center of the beam fits a one-dimensional solution. Note that the depth of the 10 W/cm3 contour
increases with increased albedo. When the albedo is equal to 0.95, all of the region above 0.035 cm has
a = 0.5
r = 0.01 cm
0
0.05
10
0.1
1.0
Depth [cm]
0.15
0.1
0.2
a
0.01
0.25
0.3 0.001
0.35
0.4
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Radius [cm]
a = 0.8
r = 0.01 cm
0
0.05
10
0.1 0.1
1.0
Depth [cm]
0.15 0.01
0.2 b
0.001
0.25
0.3
0.35
0.4
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Radius [cm]
a = 0.95
r = 0.01 cm
0
0.05
0.1 10
0.1 0.01
1.0
0.001
Depth [cm]
0.15
0.2
c
0.25
0.3
0.35
0.4
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Radius [cm]
a rate of heat generation of at least 10 W/cm3. Without scattering, the rate of heat generation at 0.035
cm for µa = 10 cm–1 is 7.0 W/cm3. This is not a trick — energy is conserved in both cases. As scattering
increases, the curve depicting the decrease in the rate of heat generation is more quickly attenuated with
depth (see Figure 2.8). As spot size is decreased (see Figures 2.6 and 2.7), radial scattering becomes
increasingly important. As a result, the region of high rate of heat generation for a beam radius of 0.01 cm
1.6
a = 0.95
1.4
Fluence rate, φ(z) [W cm-2]

a = 0.85
1.2
a = 0.5
1
(1-rs)exp(-µaz)
0.8
0.6
0.4
0.2
0
0 0.05 0.1 0.15 0.2 0.25 0.3
Depth, z [cm]
FIGURE 2.8 Fluence rate as a function of depth at r = 0 (for the data of Figure 2.5). In addition, the fluence rate
for a = 0(µs = 0) is plotted for µa = 10 cm–1.
is a depth of 0.01 cm. The penetration depths for 1 W/cm2 are about 0.15, 0.093, and 0.05 cm for albedos
of 0.5, 0.85 and 0.95, respectively.
From this analysis, it appears that scattering (as a single parameter) should not hinder coagulation or
ablation. In fact, scattering increases the maximum value of the rate of heat generation when irradiance
and the absorption coefficient are kept constant.
The inability to coagulate or ablate tissue with a Nd:YAG laser (λ = 1060 nm) is not because of the high
albedo, but is due, rather, to the low values of absorption. Often, the tissue absorption coefficient at λ =
1060 nm is less than 0.5 cm–1. In fact, over the entire spectrum from about 650 nm to 1.25 µm, µa is less
than 1.0 cm–1 for whitish-appearing tissue. To ablate whitish tissue it is necessary to increase surface
absorption with blood (especially for 1060 nm radiation) or apply an exogenous dye. For example, Indocy-
anine Green has a broad absorption peak at 780 nm. Even a thin layer of ICG (5 g/L) with an absorption
coefficient of 2200 cm–1 can produce dramatic results during diode laser radiation. Once ablation is initiated,
spots of carbonization form that provide the necessary absorption for continued ablation.
2.2.8 Selection of Wavelength

The selection of a laser should be based on the requirements for a particular task. In general, UV
wavelengths transmit through water but are absorbed by proteins and amino acids. However, the primary
absorber for wavelengths above 1.3 µm is water. Between these wavelengths, tissue chromophores such
as melanin and blood absorb the light. The influence of these absorbers as a function of wavelength is
illustrated in Figure 2.9.
The least amount of absorption of light in tissue occurs in the 600 nm to 1000 nm band of wavelengths.
The effect can be visualized by directing white light to one side of your hand. A red glow can be seen on
the other side, indicating the red (630 nm) light has propagated through the tissue while shorter wave-
lengths have been absorbed. What is seen is light that has been scattered hundreds of times. Very few, if
any, non-scattered photons are transmitted through the tissue of 1.0 cm (~ 50 optical depths). No non-
scattered photons are transmitted through several cm thickness. Nevertheless, considerable research has
been directed at using wavelengths in the 600 nm to 1000 nm window to image objects within tissue.
2.2.9 Time Resolved Propagation

Most of the imaging techniques exploit the increase in pathlength of scattered photons. Thus, for an
impulse of light incident upon tissue, the light transmitted to a point r consists of non-scattered photons
104
absorption coefficient [mm-1]

103
aortic tissue hemoglobin
102
101 water
100
10-1
excimer
10-2 holmium
argon
10-3
neodymium
10-4
10-5
0.1 1 10
wavelength [µm]
FIGURE 2.9 Absorption spectra of aorta (interrupted line), blood (dotted line) and water (solid line).
that arrive first (if there are any); this group is followed by photons scattered in a highly forward direction
such that their pathlength is not much longer than the direct path of non-scattered photons (say, 10%).
Other photons have longer pathlengths and arrive at longer times, as suggested by Figure 2.10. The
diffusion approximations suggest photon propagation to an instantaneous point source in an infinite
medium can be represented by
2
exp  ------------ – µ a ct
3⁄2 r
φ ( r, t ) = c ( 4πDct ) (2.32)
 4Dct 
where c is the speed of light in the medium, D is the diffusion constant D = 1/3(µa + µs(1 – g)) and r is
the distance from the point source.
In addition to imaging objects in turbid media, time-resolved measurements of light propagation
provide information on the absorption coefficient, µa, and the reduced scattering coefficient, µs′, where
µs ′ = µs ( 1 – g ) (2.33)
1.2
0.8
multiple scattered photons

intensity [a.u.]
0.6
0.4
0.2
_1.1r
light path <
0
0 1 2 3 4
output time [a.u.]
FIGURE 2.10 Time-resolved propagation for an impulse of light incident at r = 0 with light measurement at
position r.
The peak of Figure 2.10 is related to µs′ and the attenuation within tissue is given by µa.6
When a ray of light is delivered to a semi-infinite slab, the reflectance as a function of distance from
the center of the ray and time is6
2 –2
–3 ⁄ 2 –1 –5 ⁄ 2 r + ( µs ′ )
R ( r, t ) = ( 4πDc ) ( µs ′ ) t - exp ( – µ a ct )
exp – ------------------------- (2.34)
4Dct
For total reflectance, Equation (2.19) reduces to
–2
( µs ′ )
exp -------------- - exp [ – µ a ct ]
4Dct
R ( t ) = ----------------------------------------------------------
1⁄2 3⁄2
- (2.35)
( 4πDc ) µ s ′t
As ct becomes large, the measured attenuation provides a value of absorption.

To avoid the difficulties of time resolved measurements, it is possible to transform the diffusion
approximation to the frequency domain. Images or optical properties are evaluated by examining the
amplitude and phase resulting from irradiation with a modulated continuous wave laser.7
2.3 Thermal Response

The thermal response of tissue to laser radiation is governed by the local rate of heat generation (see
Equation (2.4)). During the initial heating phase, the temperature response prior to the diffusion of heat
from the region of heat generation is
µ a φ ( r )t
T ( r, t ) = -----------------
- (2.36)
ρc
Equation (2.4) is especially useful for estimating temperature of the end of a short pulse duration of t0.
µa
T ( r, t 0 ) = -----ψ (r) (2.37)
ρc
where ψ (r) is the fluence [J/m2] distribution of the absorbed energy. When light scattering and specular
reflection can be neglected, the temperature at t0 at the surface of tissue is
µ
T ( z = 0, r, t 0 ) = -----a W ( r ) (2.38)
ρc
where W[J/m2] is the radial profile of the radiant exposure.

In the absence of any phase changes, the transient temperature field is described by appropriate
boundary conditions and the heat conduction equation.
∂T ( r, t )
ρc ------------------ = ∇k∇T ( r, t ) + S ( r, t ) (2.39)
∂t
where r is density [kg/m3], c is specific heat [J/kg • °K] and k is thermal conductivity [W/m°K]. For an
instantaneous point source in an infinite medium, the transient temperature field is given by
2
- exp  ----------
Q 1 –r
T ( r, t ) = ----- ----------------------- (2.40)
ρc 8 ( παt ) 3 ⁄ 2  4αt 
where α = k/ρc is the thermal diffusivity [m2/s] and Q is the amount of heat instantaneously generated
at r = 0. Temperature distributions for various simple source geometries are described in Carslaw and
Jaeger8 for infinite and semi-infinite solids.
Although analytic solutions exist for Beer’s law absorption of light and a Gaussian beam profile, most
solutions of Equation (2.24) rely on either finite difference9,10 or finite element11 simulations.
Excellent agreement between measured and computed temperatures have been reported for the eye.12
However, in situations involving an air–tissue interface, Equation (2.39) has not adequately described
the thermal process even for temperatures below 100°C. Most models assure a convective boundary
condition for heat flow f [W/m2] of
f = h ( T tissue – T air ) (2.41)
where h is the convective heat transfer coefficient [W/m2 • °K]. Often, predicted and measured
temperatures do not match, as illustrated in the following example of argon laser irradiation of in
vitro human aorta.
Example
Torres et al.13,14 modeled and measured the temperature response of aorta to argon laser radiation.
The model included a finite difference solution of the diffusion approximation for light propagation.
Heat conduction was modeled with a finite difference algorithm based on Equations (2.24) and (2.26);
the rate of heat generation was based on a diffusion approximation solution for fluence rate produced
by the argon laser radiation. Optical properties were determined from spectrophotometer measure-
ments of in vitro tissue samples. Thermal properties for aorta were obtained from Valvano and
Chitsabesan.15 Temperatures were measured with a thermal camera. Measured and computed tem-
peratures at the center of the laser spot are presented in Figure 2.11. There are so many differences
between the two curves that it is difficult to know why the curves did not match. Once predicted
temperatures reached 100°C (78°C temperature rise), the temperature remained constant during a
phase of water vaporization.
Whenever experimental and predicted data do not match, many possibilities as to the source or sources
of error can be found. This example exhibits a frightening number of possibilities. The task of predicting
temperature is illustrated in Figure 2.12. Given a set of laser parameters (spot size, beam profile, irradi-
ation time, power and wavelength), and a model for light propagation, it is possible to estimate the
fluence rate in the tissue. Computing the light distribution requires knowledge of the tissue optical
properties, such as the absorption coefficient, µa, and the reduced scattering coefficient, µs′ = µs(1 – g).
80
70
60
Temperature Rise (0C)
50
40 measured
predicted
30
20
10
0
0.0 0.5 1.0 1.5 2.0
Time (sec)
FIGURE 2.11 Predicted versus measured temperature during argon laser irradiation of normal aorta with 2W beam
focused to a spot of 1.8 mm diameter. Optical properties used in the model were µa′ = 3.0 cm–1, µs′ = 30 cm–1 and
g = 0.9.
Optical Properties Absorption Coefficient Thermal Properties
Fluence rate, Heat Source, Temperature,

Laser Light φ (r, t ) Rate of S (r, t ) Heat T (r, t )
Parameters Propagation [W/m ]2 Heat Generation 3
[W/m ] Conduction [oC]
FIGURE 2.12 Block diagram of steps required in the modeling of temperature resulting from laser radiation of tissue.
Once the fluence rate has been computed, the rate of heat generation is determined using Equation (2.4).
Then Equations (2.39) and (2.41) are used to compute the transient temperature field.
Sources of prediction errors are:
• Optical or thermal governing equations do not represent the physical problem.
• The computer program does not correctly represent the governing equation (Torres et al. used a
two-dimensional, r, z, finite difference code).
• Values of the optical and thermal properties are not sufficiently accurate for the required predictions.
We have found that most users of models have complete faith in their programs — at times unrea-
sonable faith that assumes measurement differences are due to another botched experiment.
Certainly, a number of possible sources of experimental errors exist, such as:
• Inability to correctly measure a transient temperature response (in this example, a 3–5 µm Infra-
metrics model 600L thermal camera provided a two-dimensional image of surface temperature
with a new image every 33.3 ms).
• Inability to correctly measure optical or thermal properties of aorta. (Optical properties were
measured with a spectrophotometer and diffusion approximation algorithm. At 500 nm, µa = 3
to 5 cm–1 and µs′ = 30 cm–1; thermal properties of human aorta were obtained from Valvano and
Chitsabesan.15 At 35°C, k = 4.78 mW/cm • °C, α = 1.27 × 10–3 cm2/s.)
• Irradiation parameters incorrectly measured.
To determine the sources of error, it is necessary to examine Figure 2.11 and describe the major
differences. Obviously, the measured steady state temperature is far below the computed temperature,
and the initial slope of the temperature transient is about one third of the computed slope for the tissue
sample with a measured absorption coefficient of 3 cm–1. There is also a troublesome slope change at
60°C in the experimental results.
2.3.1 Error Analysis

When large differences exist between measured and predicted results, the experimental design should be
modified to simplify measurements and eliminate possible sources of measurement error. At the same
time, it may be possible to reduce the dimensionality of a problem so measurements can be compared
with an analytic solution.
In this example, Torres expanded the laser beam, so temperatures at the center of the beam would be
similar to those produced by collimated irradiation over the entire surface of a slab. The large beam
minimized the effects of radial scattering of light and radial heat conduction. At least at the center of the
beam, the optical and thermal variations were reduced to one dimension. The heat diffusion time given by
2

τ = ------- (2.42)
4α
where the characteristic length was assumed to be twice the effective penetration depth of the argon
light (588 nm, 514.5 nm) in the aorta.16
2
= ------- (2.43)
µ eff
For these conditions, τ is approximately 740 milliseconds, and significant heat conduction does not
take place for the first hundred milliseconds. On the other hand, a spot size of 1.0 mm produces a radial
diffusion time of 250 ms and significant heat conduction would take place in about 30 ms.16
During the period of minimal heat conduction, the surface temperature at the center of the laser spot
is given by Equation (2.21). Irradiation with a large spot produced a 5:1 difference between predicted
and measured temperature immediately after the onset of irradiation in Figure 2.13; the error must be
due either to the value of the absorption coefficient µa or the fluence rate φ(0). Because aorta is approx-
imately 80% water, it is difficult to believe that the thermal properties would produce such a discrepancy.
It appears the product µaφ(0) used in the model must have been too large. Independent Monte Carlo
simulations of the fluence rate agreed with the diffusion approximation results and both simulations
yielded values of φ(0) that were about 20% larger than the irradiance. Thus, it appeared that the
absorption coefficient µa had been overestimated by a factor of three.13 By reducing the absorption
coefficient (µa = 1.6 cm–1), the initial phase of the predicted and measured temperature matched.
This did not solve the difference in the steady state temperature. Predicted temperatures below 100°C
were still 25% larger than measured values. Although steady state temperature is inversely proportional
to thermal conductivity, there was no evidence that the true thermal conductivity would be a factor of
3:2 higher than the thermal conductivity measured by Valvano and Chitsabesan.15
Through additional experiments, Torres et al. determined that predicted and measured temperatures
matched for non-biological materials with known optical properties. Thus, the only source of error had
to be the governing equation or boundary conditions for heat transfer. The primary problem was surface
cooling due to vaporization of water at the surface of the tissue. This temperature-enhanced heat loss
was significant for temperatures above 60°C. A secondary heat loss was the phase change in tissue collagen
that also occurred at about 60°C.
Torres et al. achieved excellent agreement between measured and predicted temperature when the
absorption coefficient measured with a spectrophotometer was reduced and the boundary condition for
an air–tissue boundary was augmented with a heat loss due to vaporization of water.
The reader is encouraged to examine the two papers by Torres et al.13,14 that illustrate the optical-
thermal interaction of laser irradiated tissue. The single most important tissue property in laser–tissue
interaction is the absorption coefficient. Extreme care must be taken to accurately determine this param-
eter if the thermal response to laser light is modeled.
40
35 measured
30 predicted
Temperature Rise (0C)
25
20
15
10
5
0
0.0 0.5 1.0 1.5 2.0
Time (sec)
FIGURE 2.13 Predicted versus measured temperature during laser irradiation of normal aorta with 7W focused
to a 1 cm spot diameter. The absorption coefficient measured with a spectrophotometer for these samples was 5
cm–1. The effect of heat conduction can be seen in the modeled temperatures but not in the measured temperatures,
suggesting the actual characteristic length given in Equation (2.43) is less than the value obtained from the measured
optical properties. That is, the penetration depth of light was deeper than expected, owing to the overestimation of
the absorption coefficient.
2.4 Summary
Tissue response to laser radiation requires an evaluation of (1) the propagation of light in the tissue, (2)
the rate of heat generation, and (3) the resulting temperature response. However, the optical-thermal
interaction does not describe the effect on the tissue. Temperature-time-dependent rate processes describ-
ing thermal denaturation of tissue12,17 or ablation18,19 of tissue are usually the medical goals of laser
treatment.
References
1. Ishimaru, I., Wave Propagation and Scattering in Random Media, Academic, New York, 1978, Vol. 1.
2. Prahl, S.A., M.J.C. van Gemert and A.J. Welch, Determining the optical properties of turbid media
by using the adding-doubling method, Appl. Optics, 32(4):559-568, 1993.
3. Star, W., Diffusion theory of light transport, in Optical and Thermal Response of Laser Irradiated
Tissue, A.J. Welch and M.J.C. van Gemert (Eds.), Plenum, 1995, pp 131–200.
4. Keijzer, M., S.L. Jacques, S.A. Prahl, and A.J. Welch, Light distribution in artery tissue: Monte Carlo
simulations for finite-diameter laser beams, Lasers Surg. Med., 9:148-154, 1989.
5. Jacques, S.L. and L.Wang, Monte Carlo modeling of light transport in tissues, in Optical and
Thermal Response of Laser Irradiated Tissue, A.J. Welch and M.J.C. van Gemert (Eds.), Plenum Pub.
Corp., New York, 1995, pp 305-332.
6. Patterson, M.S., B. Chance and B.C. Wilson, Time resolved reflectance and transmittance for the
noninvasive measurement of tissue optical properties, Appl. Optics, 28:2331-2336, 1989.
7. Dilworth, D.S., E.M. Leith, and J.L. Lopez, Three dimensional confocal imaging of objects embed-
ded within thick diffusing media, Appl. Optics, 30:1796-1802, 1991.
8. Carslaw, H.S. and J.C. Jaeger, Conduction of Heat in Solids, Oxford University Press, Oxford, 1959.
9. Mainster, M.A. et al., Transient thermal behavior in biological systems, Bull. Math. Biophys.; 32:303-
314, 1970.
10. Incropera, F.P. and D.P. DeWitt, Fundamentals of Heat and Mass Transfer, Wiley, New York, 1985,
Chap. 6.
11. Rastegar, S. et al., A theoretical study of the effect of optical properties in laser ablation of tissue,
IEEE Trans. Biomed. Eng., 36(12):1180-1187, 1989.
12. Welch, A.J., Laser irradiation in tissue, in Heat Transfer in Medicine and Biology, A. Schitzer and
R.C. Eberhardt, Eds., Plenum, New York, 1985, pp 135-183.
13. Torres, J.H. et al., Tissue optical property measurements: overestimation of absorption coefficient
with spectrophotometric techniques, Lasers Surg. Med., 14:249-257, 1994.
14. Torres, J.H. et al., Experimental evaluation of mathematical models for predicting the thermal
response of tissue to laser irradiation, Appl. Optics, 32(4):597-606, 1993.
15. Valvano, J.W. and B. Chitsabesan, Thermal conductivity and diffusivity of arterial wall and ath-
erosclerotic plaque, Lasers Life Sci., 1:219-229, 1987.
16. van Gemert, M.J.C. and A.J. Welch, Time constants in thermal laser medicine, Lasers Surg. Med.
9:405-421, 1989.
17. Welch, A.J. et al., Definitions and overview of tissue optics, in Optical Thermal Response of Laser-
Irradiated Tissue, A.J. Welch and M.J.C. van Gemert, Eds., Plenum Press, New York, 1995, pp 15-46.
3
Optical and Thermal
Dosimetry
3.1 Introduction ......................................................................... 47

3.2 Optical Dosimetry................................................................ 47
Terms and Definitions • Light Detectors • Source Light
Measurements • On-Line Output Monitoring • In Situ
Detectors • Light Fluence Distributions in Tissue
Brian C. Wilson, Ph.D. 3.3 Thermal Dosimetry.............................................................. 69
Ontario Cancer Institute and Introduction • Thermography • Electrical Probes • Optical
University of Toronto Fiber Temperature Probes • Radiologic Imaging Methods
Douglas R. Wyman, Ph.D. 3.4 Summary............................................................................... 80
Hamilton Regional Cancer Center References ........................................................................................ 80
and McMaster University
3.1 Introduction
In many biomedical and clinical applications of lasers the accurate determination of treatment parameter
such as wavelength or delivered optical power or energy, and of the spatial and temporal distribution of
energy within laser-irradiated tissue, is essential to achieve the desired photobiological or clinical effect.
For some purposes, it is possible to rely on the built-in dosimetry systems provided by the laser equipment
manufacturer, where the user simply has to select the required treatment factors. However, as both
equipment and applications become more complex and sophisticated, it is increasingly necessary either
to check the laser system performance or to perform specialized measurements for particular treatment
techniques, for example, the local energy fluence rate on or in target tissues. The first purpose of this
chapter is to provide practical guidance in light dosimetry techniques and instrumentation. The presen-
tation will be general, rather than specific to particular clinical applications, but the principles and
approaches illustrated should enable specific measurements to be made with confidence.
The second purpose is to describe corresponding principles, techniques and instruments for deter-
mining the temperature distributions in tissue resulting from laser irradiation, because a wide range of
clinical laser-based treatments are based on thermal effects. This will include both noninvasive, or “remote
sensing” methods, based on the surface infrared emission from heated tissue, and the use of direct point
temperature measurements with microthermocouples. Thermal dosimetry will be illustrated with refer-
ence to various specific clinical and preclinical applications.
3.2 Optical Dosimetry

In this section, after first establishing the various terms and definitions relating to optical dosimetry, we
will describe the performance characteristics of the different types of light detectors that are used
0-8493-1146-2/02/$0.00+$1.50
commonly in dosimetry systems. The principles and methods for measuring laser beam parameters, with
and without optical fiber light delivery, will follow. Then, after an introduction to the characteristics of
light fluence distributions in tissue, the design and performance of optical fiber-based probes will be
described, leading to the general topic of performing interstitial measurements of light distributions in
tissue. This section will conclude with a brief discussion of recently developed, noninvasive techniques
for in situ optical dosimetry.
3.2.1 Terms and Definitions

Figure 3.1 illustrates the various laser treatment parameters for which measurement techniques will be
discussed in later sections. These include both those parameters relating to the light as delivered by the
laser or optical fiber system and those involved in the spatial or temporal distribution of light within
irradiated tissue. The corresponding terms and units are listed in Table 3.1, for both continuous wave
(CW) and pulsed irradiation.
For the laser output, delivered either as a direct beam or via optical fibers, the optical energy, E0, and
power, P0, are related simply by E0 = P0.t, where t is the treatment or exposure time. If the laser is operating
with multiple output wavelengths, the power may be given either in terms of the power at each discrete
wavelength or as the total power in all “lines.”
The power density or intensity (and corresponding energy density) is a measure of how the laser
output or light delivered to the tissue is spatially distributed. In the case of a uniform beam of area A,
the average power density is simply equal to P0/A. However, in practice, there is often a considerable
power variation across the beam. For direct optical delivery (e.g., via lenses or mirrors), the mode
structure of the laser1 is a primary determinant of the beam shape (see Figure 3.2a). The separate modes
are usually “scrambled” by optical fiber delivery, but the fiber output is not necessarily uniform and
Delivery System Tissue
Laser
. Radiance
. Fluence (rate)
. . Coupling
.Output Power
Wavelength
Efficiency
.Pulse Parameters
.
. Delivered Power
.. Fiber Loss . Spot Area

Source Distribution
. Bending
Mode Mixing
FIGURE 3.1 Elements of laser treatment. The various dosimetric parameters at each point between the laser source
and the irradiated tissue are indicated.
TABLE 3.1 Radiometric Terms and Units

Term Symbol Definition Units
Radiance Λ radiant energy flux per unit solid angle in a given direction and W.m–2.sr–1
per unit projected area perpendicular to that direction
Fluence Rate φ ∫L(ω,t)dω integral of the radiance over all solid angles at a point W.m–2
Fluence Φ ∫ φ(t)dt integral of fluence rate over the exposure time J.m–2
Each radiometric term can also be expressed as a spectral quantity by defining it per unit wavelength.
Optical and Thermal Dosimetry 49
TEM TEM TEM

00 01 02
(a) Laser
P
i
i ii
(b) Laser x ii
x
P
i
ii
x
FIGURE 3.2 (a) Examples of low-order modes of laser operation. The fundamental transverse mode (TEM00) has
a single Gaussian beam profile and is particularly suitable for direct tissue irradiation or efficient coupling into optical
fibers. (b) Beam profiles from cleaved or (micro) lens-tipped optical fibers.
depends both on how the laser beam is coupled into the fiber and whether an optical element (e.g., a
lens or diffuser) is used at the distal end (see Figure 3.2b). Note also that, in the case of a focused or
defocused beam, the power density varies with distance even if there is no loss in total power.
With a pulsed laser source (see Figure 3.3) the energy per pulse, Ep, can be determined from the
average power and number of pulses per second (pulse repetition rate, Rp) by Ep = P0/Rp. The peak
Output
Power
CW
Po Pp
Ep
time τ
(a) (c)
Pulsed
Pp
Pp Pp
/e Ep
time τ
(b) (d)
FIGURE 3.3 Illustrations of the output power time dependence for (a) continuous wave (CW) and (b) pulsed
sources, and the detailed pulsed shapes for (c) square-wave pulses and (d) Gaussian pulses.
power in the pulse, Pp depends on the pulse shape and, if present, substructure. In the simplest case of
a square pulse of length (duration) τ, such as produced by a mechanical chopper or acousto-optic
modulator, Pp = Ep/τ. For the more common (approximately) Gaussian pulse of width τ (full width at
1/e or 37% of the peak power), Pp = 0.886 Ep/τ. If there is substructure, for example, multiple
micropulses within each main pulse (macropulse), then its characteristics must be known in detail and,
depending on the biophysical effects induced in the tissue, the relevant dosimetric quantity may be
either the peak power and energy in each micropulse or in the whole macropulse. The power and energy
densities for pulsed irradiation are determined from the geometric characteristics of the beam in the
same way as for CW sources.
With the exception of the transparent elements of the eye (cornea, lens, vitreous), light is strongly
scattered upon entering tissue, due to microscopic variations in the tissue refractive index.2,3 Generally,
this does not result in energy loss, but the energy is spatially (and also, with short pulses in the sub-
nanosecond range, temporally) redistributed. Before or after multiple scattering, the light may be
absorbed by tissue chromophores (endogenous or exogenous), resulting in the photobiological effect, or
be lost to the tissue volume as reflected or transmitted light. Secondary photons (of longer wavelength)
may be produced following absorption, which may, in turn, be scattered, reabsorbed or escape from the
tissue.4 These absorption and scattering processes vary strongly with wavelength and tissue type.
As illustrated in Figure 3.4, usually the energy flow at any point, x, within the, tissue is not equal in all
directions, so that the angular distribution of the local power (energy) density is described by the radiance,
L(x, Ω), which is the power density per unit solid angle, Ω. The total energy fluence rate, φ, at this point,
is equal to the radiance integrated over all solid angles, and represents the total energy flow per second
per unit cross-sectional area through the point. (The local energy absorption rate and consequent pho-
tobiological effects are generally insensitive to differences in the radiance distributions, but the latter do
affect light dosimetry measurements: see Section 3.2.4.) At low fluence rates, the local rate of energy
absorption equals the product of the tissue absorption coefficient (probability of absorption per unit path
length of photon travel) and the local energy fluence rate. However, at high incident power densities, such
as with short pulse, high-peak-power lasers (≥ 106 W.cm–2), non-linear effects such as multi-photon
absorption may occur, where the energy absorption rate depends on some power function of the local
Incident
Light
L(x~,Ω)
x Tissue
~
Local Fluence Rate

φ(x
~)= ΩL(x
~,Ω)
Local Energy Absorption Rate
. ~)
~) φ(x
=µa(x
FIGURE 3.4 Illustration of a non-isotropic radiance pattern of light at a point within tissue. The contribution to
the local fluence (rate) is not equal from all directions, due to the combination of the primary (unscattered) light
and the scattered flux.
fluence rate (e.g. ∝ φ2 for 2-photon absorption). This chapter will not be concerned with the details of
these absorption processes and subsequent biological effects,5 except insofar as they influence the types of
physical dosimetry measurements required to characterize the light irradiation or resulting temperature
distributions.
3.2.2 Light Detectors

All the dosimetric techniques described below rely on suitable detectors to convert the optical energy
into electrical signals, which can be measured by standard electronic instruments.6,7 Alternative “dosim-
eters,” for example, actinometers based on the measurement of photochemical changes,8 will not be
considered bacause they are not in common use. The selection of a particular detector for a specific
application depends on matching the detector response (spectral range, sensitivity, response time, etc.)
to the expected optical conditions. However, there are also significant differences in factors such as ease
of use and cost, and one must also consider whether a particular instrument is for a single use or for a
range of potential applications in different light fields.
3.2.2.1 Photothermal Detectors
Figure 3.5 shows a detector system based on the absorption of incident light by a “blackbody” surface
(i.e., close to 100% absorption) and the measurement of the resulting temperature rise of the surface by
conversion to an electrical signal. The main advantages of such detectors is that they have an almost flat
spectral response over a very wide wavelength range, do not depend on the pulse characteristics of the
incident light, and can be used up to high power levels. Conversely, they have a slow time response (which
limits their use if the light varies over a comparable time scale) and relatively poor sensitivity. They are,
therefore, most often used for measurements of the average power output of lasers. The sensitive element
may be of different size and shape for different applications, and the detector “heads” are often provided
on a cable remote from the read-out instrument, which gives flexibility in positioning the detector.
3.2.2.2 Photodiode Detectors
Photodiodes are solid-state semiconductor devices in which the absorption of incident photons causes
the production of free electron-hole pairs, which are subsequently detected as an induced electrical
current. The efficiency of this process is strongly wavelength dependent and depends also on the photo-
diode material, so that any one type of photodiode has a limited spectral range, and even within this
range, the sensitivity is wavelength dependent, as illustrated in Figure 3.6a. However, within their ranges,
the sensitivity of photodiodes is high and they have a good time response for many laser types and
applications. The performance of photodiodes does, however, vary with the quality (and hence cost) of
the device and associated electronics. For clinical dosimetry, high sensitivity, general-purpose radiometers
using photodiodes are available (see Figure 3.7), with a range of detachable detectors of different spectral
ranges, sensitivities and geometric forms, and with both power and energy registration. These may be
bench-top or hand-held configuration. They cost from a few hundred to a few thousand dollars and are
generally rugged and reliable. Depending on the manufacturer, they are either available with a built-in
wavelength calibration or this is supplied in tabular or graphic form. Auto ranging is often provided for
ease of use over a wide range of detected powers. If absolute light dosimetry is important, then it is
advisable to ensure that the calibration can be traced back to a national standards laboratory. The upper
power level is limited, both by potential damage to the photodiode itself and, more usually, by the built-
in range of the electronics.
3.2.2.3 Photomultiplier Detectors
Photomultipliers are vacuum-tube devices in which electrons released from a light-sensitive surface
(photocathode) are electrostatically accelerated by a series of electrodes at increasing voltage, causing the
release of further electrons, resulting in a highly amplified electrical pulse. The spectral range is also
limited for these devices (see Figure 3.6b). They are relatively expensive and fragile and require special
power supplies and detection electronics. Their advantages are very fast response time and extremely
FIGURE 3.5 Example of a photothermal power meter, based on light absorption by a blackbody surface and
measurement of the consequent temperature rise. Such instruments typically have a range of interchangeable detector
“heads” with either surface or volume detector elements. Different power ranges and laser types may be selected.
Electronic time integration for energy measurements may also be provided.
1.0
Relative Response
0.8
In Ga As Bialkali
Si
0.6 Sb-Cs
0.4
Multialkali
0.2
0.0
400 800 1200 1600 2000 400 800 1200 1600 2000
Wavelength (nm) Wavelength (nm)

(a) (b)
FIGURE 3.6 (a) Typical spectral response of silicon and indium gallium arsenide detectors, suitable for measure-
ments at visible and near-infrared wavelengths. (b) Typical spectral responses of photomultiplier tubes of different
materials.
high sensitivity (low noise equivalent power) within their operating wavelength range, the latter resulting
from the high signal gain within the tube itself. They can be operated in “current mode,” where the pulses
from many incident photons are integrated as an electrical current, or in “single photon counting mode,”
where individual pulses are detected digitally. In this mode, PMTs have typically about a 10% quantum
efficiency (i.e., one photon in ten is detected), and the sensitivity is limited only by the spontaneous
emission of electrons from the photocathode. This can be minimized by cooling the tube (either elec-
trically or with liquid N2) and, in a practical biomedical setting, a dark level of about 5–10 photons per
minute can be achieved, which is equivalent to a CW power of about 10–19 W. In this case, the upper
limit on incident power is set by the electronics, typically ~107 counts per second (~10–12 W). Thus, the
price paid for extremely high sensitivity is a restricted power range. The balance can be shifted toward
FIGURE 3.7 Example of a general-purpose, hand-held photodiode radiometer. These are available with a variety
of detector head sizes and geometries for different applications.
higher powers by operating in current mode. Recently, solid state “avalanche photodiodes” have become
available that also have intrinsic signal amplification, giving sensitivities on the order of those of some
photomultiplier tubes. These have the added advantages of small size and ruggedness.
3.2.2.4 Accessory Devices
Several items of equipment are useful accessories to the main detectors for practical dosimetry. Three of
these are:
1. Filters and monochromators — A wide range of spectral filters is available to select light only
within a defined wavelength range.10 As illustrated in Figure 3.8, these may be band-pass, cut-on
(high pass) or cut-off (low pass) depending, respectively, on whether light only within a defined
band, above a given range, or below a given range is required. If scanning of a wide wavelength
range is needed, the light can be passed through a monochromator placed in front of the detector.
If very strong rejection of unwanted wavelengths is required (e.g., in measuring tissue fluorescence
on a background of ambient or scattered light), filters or monochromators can be “stacked,” e.g.,
two band-pass filters, each with 50% transmission at the peak and 10-4 transmission out of the
band will, when used together, give 25% peak transmission and a 108 rejection factor for unwanted
light. Note that there is always some loss at the selected wavelength, so that this must be taken
into account for absolute dosimetry measurements. The loss through a monochromator depends
on the design, slit width used and divergence or convergence of the incident light, so that it is not
simple to determine these factors. Neutral density filters, which attenuate light over a very wide
wavelength range, are useful for reducing the light intensity onto the detector to stay within the
correct operating range. In most wavelength ranges, ND filters have a rather flat transmission
spectrum. The attenuation is usually cited in optical density units: OD = log10(Io/It), where Io =
incident intensity and It = transmitted intensity. Thus, for example, filters of OD = 1, 2, 3 have
attenuation factors of 10, 100, and 1000, respectively. For two or more filters used in combination,
the ODs are added (attenuation factors multiplied).
T(λ)
T(λ)
lo(λ)
Cut Off l(λ) l(λ)
Band-Pass
T(λ)=
1 Cut On lo(λ)
T1 T2
lo(λ) l(λ)
l(λ)=lo(λ).T1(λ).T2(λ)
T12(λ)=T1(λ).T2(λ)
FIGURE 3.8 Spectral transmissivities of different types of wavelength filters. Note that the maximum transmission
is always less than 100%, and that there is some residual “leakage” at wavelengths outside the intended transmission
range. For stacked filters, as illustrated on the right, the spectral transmission at each wavelength is the product of
the individual transmissions.
2. Integrating spheres — Most photodetectors have a flat sensitive surface. This is suitable for
measuring direct light beams where all, or a known fraction, of the beam area can be made normal
to the detector surface. However, in many cases, particularly when optical fibers are used, either
for light delivery or collection, the light is distributed in many directions and not necessarily
uniformly. As illustrated in Figure 3.9, an integrating sphere can be used to collect all the light,
regardless of direction, so that, after being “randomized” by multiple scattering from the inner
diffusely reflecting surface, a known fraction of the light is detected. The larger the sphere, the
simpler it becomes to couple the light into it, but the lower its sensitivity, because the fraction of
light ultimately falling on the detector depends on the ratio of detector area to surface area of the
sphere. For a 2–5 cm diameter sphere with a 1 cm diameter detector port, the efficiency (light
detected or light input) is typically about 1%. A baffle is often used to shield the detector from
direct light from the source, thus reducing further the dependence of the signal on source position
and geometry.
3. Beam choppers — It is often convenient to “chop” a CW beam to provide (square wave) pulses
of light. The magnitude of the change in detected signal can then be separated from any constant
background light (see Figure 3.10). This increases the detection sensitivity, eliminates effects due
to drift and, most importantly, permits measurements to be made under ambient room lighting.
Simple and inexpensive mechanical choppers, operating up to a few kHz, are available. (Faster
chopping can be obtained by using an acousto-optic modulator, but these are more expensive and
complex to operate.) The detector output signal can be measured with an oscilloscope or, more
accurately, using a lock-in amplifier synchronized with the chopper.
3.2.3 Source Light Measurements

This section examines issues in the measurement of laser light sources, either using the direct laser beam
or where optical fibers are used for light delivery to the patient. It will be assumed that the appropriate
detector system has been selected, based on matching the sensitivity, spectral range and temporal response
to the source operating conditions.
Optical and Thermal Dosimetry
FIGURE 3.9 Illustration of the principle of an integrating sphere. The detected signal is approximately independent of the source geometry, since all photons are multiply
scattered by the inner surface before reaching the detector. The baffle prevents the detector from “seeing” the source directly.
55
Po Po
S
B Detector
t t Scope
S
B
Lock-In
Driver S
Amplifier
Chopper
FIGURE 3.10 Principle of the use of an optical chopper to measure light signals in an ambient light background.
The chopped signal is shown either measured with an oscilloscope or via a lock-in amplifier (LIA) in which the
constant baseline offset is subtracted.
3.2.3.1 Direct Beam Sources

Figure 3.11 shows the two main conditions for direct beam measurements (with or without optical
elements such as lenses, mirrors or filters between the laser and the detector). In the first case, the beam
is smaller than the detector-sensitive area. Thus, the optical power can be determined directly if the
detector calibration is known at the source wavelength. In the second case, where the beam size at the
detector is greater than the detector area, the signal at any position in the beam (after response calibration)
is equal to the average power, P, over the detector area, a, i.e., to the local beam intensity (P/a). If the
intensity is uniform, then the total power, Po = P . (A/a), where A is the cross-sectional area of the beam.
If it is not uniform, as, for example, in the case of the output from a flat-cut optical fiber, then the total
power can be obtained either by using an integrating sphere, as indicated above, or by integrating the
local intensity measurements over all points in the beam. In most cases, the beam will be radially
symmetrical, so that a single profile across a beam diameter suffices.
3.2.3.2 Optical Fiber Sources
For a flat-cut fiber source, the beam profile can be obtained as described above. For fibers having a light
diffusing tip,11 it is easiest to use an integrating sphere (as illustrated in Figure 3.9) to measure the total
output power, making sure the sphere is large enough to encompass the complete diffusing tip. If it is
required to determine the spatial distribution of the fiber output, for example, the isotropy of a point
diffuser or the profile along the length of a line diffuser, then a small flat-face detector can be scanned
around or along the tip, ensuring that the detector is always oriented normally to the fiber and at a
constant distance. The total power can be determined in this way, by integrating over the surface area
of, for example, a sphere traced by the detector at a constant distance. However, this procedure is time
consuming and, unless a precision scanning mount is used for the detector and the complete surface is
scanned, it is generally more accurate and convenient to use a calibrated integrating sphere.
Po
(a) (b)
FIGURE 3.11 Illustration of total beam power measurement for the cases of (a) beam size less than detector, (b)
large beam, showing the measured signal profile from which the total power may be obtained by area integration.
A qualitative estimate of the output distribution from a fiber source can be obtained by placing the
source within a light-scattering medium (e.g., dilute milk). The output can then be visualized directly.
A fluorescent liquid, excited at the source wavelength, can also be used for this purpose, imaging the
emitted light. Some trial and error with dilutions and placement of the source are usually needed to
obtain a satisfactory picture in this way. It is possible to make this technique semi-quantitative, for
example, by using a video or CCD camera to image the source.12
3.2.3.3 Spectral Measurements
The output spectrum of a light source can be determined using either a monochromator or set of pass-
band filters. The former gives a continuous wavelength scan with a spectral resolution determined by
the bandwidth of the monochromator (typically a few nm for a simple laboratory instrument), while
the latter samples the source output spectrum only at discrete wavelengths. More sophisticated and rapid
systems (optical multichannel analyzer) use a diffraction grating spectrograph to spread the spectrum
onto either a photodiode array or charged-coupled device (CCD). This can be particularly useful for
measuring, for example, fluorescence spectra in tissue.
In cases where the source spectrum is broad band, it may be necessary to correct the measurements
for the spectral response of the detector at each wavelength. If the measured power (density) at wavelength,
λ, is Pm(λ) and the corresponding detector responsivity is R(λ), then the true source power is
P t(λ) = Pm(λ)/R(λ) (3.1a)
The total output power, integrating over all wavelengths is then
Po = ∫ P t(λ) dλ = ∫Pm(λ)/R(λ) dλ (3.1b)
3.2.3.4 On-Line Output Monitoring

It is often useful during a laser treatment to monitor the power delivered to the tissue in order, for
example, to detect output variation. Also, many in vitro or in vivo spectroscopic measurements require
To Monitor Treatment
Reflected Light Detector Beam
Fiber
Laser
Lens To Monitor
Glass Slide Detector
To Tissue/
Delivery System
(a) (b)
FIGURE 3.12 Techniques for monitoring laser source output power, (a) in a direct beam, by detecting the specular
reflection from a glass slide and, (b) with fiber coupling, by measuring the leakage component.
the delivered power to be known throughout the measurements. With clinical laser equipment, this on-
line monitoring is often built into the system and used actively to control the output.
When an open beam is used, the simplest method, as shown in Figure 3.12a, is to pass the beam
through a thin glass slide and detect the small percentage of light reflected. The slight loss in primary
beam power can easily be determined by measuring the transmitted beam with and without the slide.
One potential problem with this method is that the fraction reflected depends on the polarization of the
beam, which, in some lasers, may drift with time.
With optical fiber delivery, where the coupling efficiency into the fiber may be variable and it is not
adequate simply to check the delivered power at the start and end of treatment, measuring the leakage
from the fiber can be a useful way to check at least that the output is not varying during treatment (see
Figure 3.12b). In this case, because the leakage depends on the bending radius of the fiber, it is necessary
to hold the fiber straight and at a fixed position with respect to the detector by, for example, using a
grooved clamp around the fiber, to which the detector attaches. Even then, the method is not easily
amenable to making absolute, reproducible measurements.
3.2.4 In Situ Detectors

For measurements of surface, intracavitary or interstitial fluence (rates), as discussed in Section 3.2.5,
specifically designed detectors are usually required. Factors to be considered include safety, sterilizability,
size, spatial and spectral response, degree of invasiveness and suitability for intraoperative, endoscopic
or interstitial use. In some cases, small photodiodes held or sutured onto the tissue surface have been
used directly.13 Electrical safety and restricted options for sterilization are limitations, and generally, the
size of these detectors precludes their being used endoscopically or interstitially. On the other hand,
calibration for absolute fluence rate measurements is relatively straightforward.
For interstitial and endoscopic measurements, optical fiber detectors offer many advantages, at least
in the visible and near-infrared wavelength ranges where a variety of types of fiber with very low
transmission losses and very small diameter are available. These can be easily coupled to appropriate
photodetectors, depending on the required sensitivity, spectral and temporal response.
If the fluence to be measured is approximately isotropic, as in deep within a scattering tissue, then the
distal end of the fiber may simply be cleaved flat, as illustrated in Figure 3.13a. The orientation of the
fiber tip does not alter the reading in this case, and the numerical aperture of the fiber in the tissue
determines what fraction of the light incident on the cleaved face will enter the fiber and be detected.
The detection sensitivity is proportional to the cross-sectional area of the fiber core.
In an anisotropic light field, for example near an irradiated tissue surface or implanted (optical fiber)
source, the response of a flat-cut detector fiber depends on its orientation. It is possible to determine the
fluence rate at a point approximately by measuring at several orientations, as shown in Figure 3.13b, and
Tissue Tissue
(a) (b)
FIGURE 3.13 Interstitial measurement of the local fluence (rate) using cleaved optical fiber detectors: (a)(approxi-
mately) isotropic field using a single detector at an arbitrary orientation to the light beam, (b) anisotropic field with
measurements made simultaneously or sequentially at several specific orientations.
summing up the readings after appropriate weighting for the solid angle acceptance in each orientation.14
However, this is tedious and highly invasive, especially if the fluence is required at multiple points in the tissue.
Two solutions aimed at making detector fiber tips with good isotropic response so that the orientation
does not matter, even in an anisotropic field, have been developed. The first15 is to make the tip spherical
and highly light scattering so that photons entering it from any direction are randomized and have the
same probability of being transmitted to the photodetector (Figure 3.14a). The second type of isotropic
detector16 is based on making the tip fluorescent (Figure 3.14b). The emitted fluorescent light is intrin-
sically isotropic and the tip geometry can be adjusted to make the response independent of the direction
of the excitation light.
The advantages of scattering-tip detectors are their relatively high sensitivity (typically ~10–5 –10–4
cm2)16 and the fact that the spectral response is essentially set only by the photodetector used. Their
principal limitation is that they cannot be made arbitrarily small, because the isotropy is lost if the optical
pathlength in the tip is inadequate to give multiple scattering; commercially available isotropic probes
have tip diameters of about 800 µm. The isotropy of fluorescent-tipped detectors is independent of size,
provided the tip shape is maintained. However, any given detector has a limited wavelength range, and
an additional pass-band or cut-on filter is required to discriminate against the excitation light (the direct
detection of which is not isotropic). For a given tip diameter, the sensitivity is typically a thousand-fold
less than the equivalent scattering tip, but this can be compensated for, at least in the visible range, by
using more sensitive detection schemes, such as single photon counting. Fluorescent tips with good
isotropy (± 15% except in the 20º backward angle of the fiber stem) down to diameters of 170 µm (which
will pass down a 26 G needle) have been reported,16 although these are not currently available commer-
cially. Both types of detector fiber can be sterilized for clinical use. The useful life of the fluorescent tips
is limited by fluorophore bleaching in some cases. The use of fluorescent probes with pulsed irradiation
can also be limited by the fluorescence decay time of the tip (~ns). The response time using scattering
tips is that of the photodetector, unless sub-picosecond pulses are involved.
The calibration of optical fiber detectors for in situ measurements in tissue involves numerous factors,
particularly if absolute values of the local fluence (rate) are required.17–19 For relative measurements with
a single detector, for example, monitoring with time during a treatment or at different positions in the
radiation field, calibration is unnecessary. For relative measurements between several detectors, it is
sufficient to measure the response of each in the same (arbitrary) field. It is, however, important to have
the correct refractive index match (or mismatch) as in the intended treatment, because the detector
λ λem
λ λex
Cut-On
To Detector
Filter
Tissue Tissue
λem
λex
(a) (b)
FIGURE 3.14 Isotropic fluence detectors based on (a) light scattering in a spherical diffusing tip and (b) fluorescence
excitation in the tip, with a filter to remove the scattered excitation light that enters the fiber.
isotropy and response depend on this and the effect is not, a priori, the same for each detector; for
interstitial applications, it is convenient to use a broad (uniform) beam and a water tank for the relative
calibration. If multiple fibers are to be used sequentially with a single photodetector, it is also important
to ensure the reproducibility of coupling between the fiber and detector.
The most accurate method to perform an absolute response calibration of an optical fiber detector
is to measure the response at depth in a medium of known light-scattering and -absorbing properties
and refractive index, which approximate those of tissue.19 The medium, which should be large in
volume so as to avoid boundary effects, is illuminated by a broad beam of known incident intensity.
(Alternatively, an optical fiber source of known output power can be used and the detector placed at
a known distance from this.) A suitable model of light propagation in tissue (see Section 3.2.5) is then
used to calculate the absolute fluence rate, φ, at the position of the detector tip, so that the detector
responsivity (at given wavelength) is R = S/φ, where S is the measured signal. In this form, the
responsivity includes the photodetector response as well as the optical fiber efficiency, but the former
can be removed if the photodetector is itself calibrated and the coupling efficiency to the detector fiber
is known.
3.2.5 Light Fluence Distributions in Tissue

In this section, the general characteristics of light fluence distributions in tissue and their dependence
on tissue optical properties are discussed, together with practical methods to estimate or measure these
distributions in vivo, including the use of the optical fiber probes presented above.
3.2.5.1 Homogeneous Tissues
The simplest case is the fluence distribution in tissues that are optically homogenous, i.e., have approx-
imately the same optical properties throughout the irradiated volume. For an external beam irradiance
of incident fluence rate φ0 [W . m-2] the primary fluence from the source is attenuated due to the combined
scatter and absorption processes. Thus, at depth d in tissue, the primary fluence rate, φp, is given by
φ p = φ 0 . exp( -µt.d) (3.2)
where the total attenuation coefficient µt = µa + µs and µa, µs are the absorption and scattering coefficients,
respectively. Note that these coefficients, which describe the probabilities of each type of interaction,
depend on the tissue and on the wavelength2–5 being the result of the chromophore content of the tissue
(absorption) and the microscopic variations in refractive index (scattering). In cases where absorption
dominates (e.g., at UV and mid- to far-infrared wavelengths), the total fluence is simply equal to the
primary fluence, which falls with depth as exp(–µ a. d), being a maximum (φ0) at the surface
(Figure 3.15a).
If the scattering is significant (as in the case of visible and near-infrared wavelengths in soft tissues,
with the exception of the transparent elements of the eye), then the fluence rate within the tissue has a
contribution from secondary, or scattered, photons. Thus, the total fluence rate is
φ(d) = φp(d) + φs(d) (3.3a)
For very high scatter-to-absorption ratio, this can be expressed analytically to a good approximation
using diffusion theory20 as
φ(d) = A.φ0.exp(–µ eff.d) – B⋅ φ 0.exp(–µ't.d) (3.3b)
The transport attenuation coefficient, µt = µa + µ's. = µ a + µ s (l–g), where g is the scattering anisotropy.
(g = 0 for isotropic scatter, →1 for forward-peaked scatter.) For large incident beam size (≥5 – 10/µ's)
the effective attenuation coefficient can be written in terms of the absorption and transport scattering
as
µeff ≅[3 µ a (µ a + µ's)]1/2 (3.4)
In Equation 3.3b, A and B are positive constants that depend on µ a, µ's and the relative refractive index
at the tissue surface. At large depth, d >>1/µ eff, φ can be written as
φ(d) = ks.φ o.exp(–µ eff.d) (3.5)
where the “backscatter” factor, ks (≡A), increases with increasing tissue albedo (µ s/µ t).
The consequences of high light scattering are seen in Figure 3.15b:
(a) The fluence rate within the tissue near the surface is greater than the incident fluence rate due to
the backscattered photons.
(b) There is sideways spreading of the beam profile.
(c) The fall-off with depth depends on the scattering and absorption properties but is less rapid than
that of the primary fluence alone.
(d) Backscattered light is lost through the irradiated surface as diffuse reflectance, Rd.
(e) The shape of the sub-surface distribution depends on the refractive index match or mismatch at
the surface (e.g., water or air).
Table 3.2 gives values of optical properties for some typical soft tissues and wavelengths, and also the
1/e depth, i.e., the depth over which the fluence rate falls to 37% of its value (δ = 1/µ eff ). For long
wavelengths (≥1500 nm) the attenuation of soft tissues is dominated by water absorption. In the near
infrared (~700–1300 nm), the absorption of other chromophores and of water is low and the tissue is
scatter-dominated, giving the maximum depth of penetration of the light (“optical window” or “thera-
peutic window”). In the visible and UV, the wavelength dependence of absorption is very complex, due
to the contribution of several types of endogenous chromophores, each with a distinct absorption
spectrum (hemoglobin, melanin, cytochromes in the visible; nucleic acids, proteins and water in the
UV). The scattering falls gradually with increasing wavelength and, in the visible range, a mixed scattering
and absorption situation prevails. The attenuation is very sensitive to differences in tissue pigmentation,
blood content and oxygenation in the visible and near infrared. In the UV, absorption again dominates,
and differences among tissues are smaller.
φo Rd
φo
φ φ
Increasing
Radiance
φo k sφ o Isotropy
-µ d In φ
In φ e a φo -µ d
e eff
-µtd
e
d d
(a) (b)
Tissue Tissue
e-µar
2
In(r φ) In(rφ) e-µeffr
r r
(c) (d)
FIGURE 3.15 Fluence (rate) distributions for external (laser) beam irradiation (a,b) or interstitial irradiation (c,d)
for the cases of: (a,c) purely absorbing tissue, (c,d) scattering and absorbing tissue.
TABLE 3.2 Typical Optical Properties of Soft Tissues

Tissue λ µa (cm–1) µs (cm–1) g µeff (cm–1) δ Rd (%)
<300 nm
Any soft 102–104 ~103 < 10 µm
(excimer)
Lightly pigmented ~1 5–10 ≥1 mm 10–20
488/514 nm
Highly pigmented ~10 102–103 ≥0.95 >10 ≤1 mm <10
(argon)
Blood ~10
Lightly pigmented <1 1–10 1–10 mm 20–50
633 nm ~102 ≥0.95
Highly pigmented <10 ~10 ~1 mm 10–20
(HeNe, dye)
Blood ~5 ~103 >0.97
1064 nm
Any soft 0.5–5 ~10 2 ≥0.9 1–5 2–10 mm 20–50
(Nd:YAG)
~2 µm
~102 <50 ~102 ~100 µm
(Er:YAG:Ho)
2.94 µm
Any soft ~3.103 ~3.103 ~3 µm
[H2O max]
10.6 µm
~103 ~103 ~10 µm
(CO2)
Note: The values should be considered as representing typical ranges in each case for general catagories of mammalian
soft tissues [5, 10, 21, 22 and refs. therein]. The values for blood are for whole, oxygenated blood at 40% hematocrit.
For absorption-dominated cases, the 1/e penetration depth is taken as d = 1/µa; for high scattering cases (µs >>
µa) as d~1/µeff.
With interstitial irradiation, e.g., from an implanted optical fiber source, the fluence distribution
around the fiber is dependent on both the absorption and scattering properties of the tissue at the
irradiation wavelength and the geometry of the source fiber tip (cut-end, isotropic diffuser, cylindrical
diffuser).11,23 In the simplest case of an isotropic (point) source of tip radius delivering an optical power
po, the fluence rate at radial distance, r, is given by:
absorption dominated: φ(r) = P0 . [exp–(µ a . (r-a))]/4πr2 for r>a (3.6a)
scatter dominated: φ(r) = P0 . ([µ2 eff./µ a)exp–(µ eff . (r-a))]/4πr2 for r>a (3.6b)
Note that, when the tissue is primarily absorbing, the geometric factor describing the spherical spreading
of the light varies as 1/r2, whereas, with high scattering, this has a weaker l/r dependence due to light
scattered back toward the source (see Figure 3.15c and d). In both cases, the exponential attenuation (µa.
or µeff ) dominates over the geometric spreading at large distances.
The biological and resulting clinical consequences of the form of the fluence distribution in tissue
depend very strongly on the tissue type, wavelength and irradiation geometry. This will be clear in the
other chapters dealing with specific applications of different laser types. However, to indicate the extremes,
the low scattering and very high absorption at UV and mid- and far-IR wavelengths means that tissues
may be cut or ablated with little spreading of the light beyond the deposition zone (depth ~ δ). Conversely,
with near-IR irradiation, the scattering causes energy absorption throughout a large volume of tissue,
going beyond the geometric edge of the beam. The behavior in the visible range is intermediate and very
strongly wavelength dependent; for example, there is a significant difference in blood absorption between,
say, an argon ion laser at 488/514 nm and a dye laser operating at 575/585 nm, the latter corresponding
to an oxyhemoglobin absorption peak. This causes a profound difference in the confinement of laser-
induced heat damage to blood vessels, as in treatment of port-wine stain.
3.2.5.2 Inhomogeneous Tissue
At wavelengths where absorption (due to water for mid-IR or cellular biomolecules for UV) dominates,
tissues can generally be considered optically homogenous unless, for example, there are significant
variations in hydration state. However, in the near-IR and, especially, visible ranges, local variations in
sc (i)
e
(ii)
d
(a)
b
d e d b d
Depth
(i)
φ
(b)
(ii)
Depth
FIGURE 3.16 Idealized representation of the fluence–depth distribution (i) and local rate of energy absorption (ii)
in: (a) a skin layer model (sc: stratum corneum, e: epidermis, b: blood plexus, d: dermis) at wavelengths with high
blood absorption. eµa > dµa << bµa, (b) a “hot center” model with discretely localized high absorbers.
chromophore content can markedly alter the distribution of fluence and of absorbed energy deposition
within the tissue. Two classes of tissue distribution have been considered; layered tissues and localized
absorbing centers. The former is particularly relevant to skin irradiation,24 and the latter has been applied
to the spatial confinement of energy in melanin granules.25
An example of the fluence rate, and resultant energy deposition rate, of visible light in a simplified
model of the skin is shown in Figure 3.16a. The blood layer, in particular, affects the fluence distribution,
reducing the fluence rate to deeper lying tissue and, at the same time, absorbing a significant proportion
of the light energy incident on it. In the case of localized absorbing centers, if the volume concentration
of these is small, the effect on the light distribution may be slight (and approximated by assuming that
the same total absorption is uniformly distributed); however, the rate of energy absorption within the
centers themselves may be very high compared with that of the surrounding tissue structure (see
Figure 3.16b), resulting in high local temperatures.
In each case, the temperature reached by the highly absorbing elements depends on the local fluence
rate, their absorption coefficient and volume and the rate of heat dissipation. This will vary markedly
with laser wavelength and pulse characteristics, with shorter pulses helping to “confine” the heat to the
local absorber.26
3.2.5.3 In Situ Determination of Fluence Rate Distributions

Two general approaches to the determination of light fluence (rate) distributions in laser irradiated
target tissues are direct interstitial measurement and calculation based on knowledge of the tissue optical
properties and irradiation geometry.
1. Direct Measurements — Direct in situ measurement of the fluence rate at discrete points within
tissue can be made using the probes described in Section 3.2.4.27,28 Isotropic probes placed inter-
stitially will measure the total fluence rates (Figure 3.17a). The use of such probes on a tissue
Tissue Tissue
(a) (b)
Tissue Tissue
(c) (d)
FIGURE 3.17 Direct in situ measurement of local fluence (rate) in tissue using isotropic optical fiber detectors or
photodiodes: (a) interstitial measurement of total fluence rate, (b) measurement of surface fluence (primary +
backscattered), (c,d) measurement of primary incident fluence, using (c) a small opaque shield to block the back-
scattered light or (d) a photodiode with the sensitive surface toward the beam. The backscatter can be monitored by
switching the probe and shield positions in (c) or placing the sensitive photodiode face on the tissue (reverse of (d)).
surface in air (Figure 3.17b) may be uncertain by up to 30 or 40%, due to the refractive index
mismatch between the probe and the tissue–air interface. If the primary incident fluence from
surface irradiation is to be measured, then the sensitive tip should be shielded from backscattered
light (Figure 3.17c). This is usually intrinsically the case if a flat photodiode is placed “face up”
on the tissue. The detector (± shield) should be small compared with the incident beam diameter
so as not to distort the fluence distribution significantly.
The diffuse reflectance, Rd, can be measured using an integrating sphere on the tissue surface29
with narrow beam irradiation (Figure 3.18a), or by using a lens or optical fiber (bundle) to transfer
or image30 a fraction of the light onto a detector (Figure 3.18b). In either case, Rd should be
measured with reference to a calibrated diffuse reflectance standard, which comprises a flat surface
of a highly reflecting material such as barium sulfate.
The accuracy of direct fluence measurement using interstitial probes is, of course, limited by
the size and positional accuracy of the sensitive tip. This can be a particular problem in hetero-
geneous tissues, such as the skin, where the depth profile of the fluence depends strongly on the
specific thicknesses and optical attenuation of the different layers. To date, such direct microdo-
simetry studies have been very limited.16
A further problem with interstitial measurements, at least in vivo, is the possible error caused
by bleeding around the tip due to the mechanical trauma of introducing the probe. This can
significantly reduce the detected signal, particularly at visible wavelengths where hemoglobin
absorption is high.
PR
Tissue Tissue
(a) (b)
FIGURE 3.18 Measurement of total diffuse reflectance, Rd, using: (a) an integrating sphere in contact with the tissue
surface, (b) a non-contact camera with a collector lens at a fixed distance from the surface and incident source
position. In each case, a fixed fraction of the diffusely reflected light is detected, determined from equivalent
measurements on a calibrated reflectance standard.
2. Calculation — If the optical properties (µa, µs, g, refractive index) of the tissue are known at the
treatment wavelength, then the complete fluence distribution can be calculated for a given irra-
diation geometry.3,11,20 For absorption-dominated cases, this is simple, as seen above. In the
scattering-dominated case, at least for simple irradiation geometry and homogenous tissue vol-
umes, the fluence distribution can be calculated analytically using diffusion theory.31 In situations
where this breaks down (e.g., if the scattering is not high compared with the absorption, or near
sources and boundaries) or is too cumbersome to apply (e.g., for a heterogeneous tissue), numer-
ical models such as Monte Carlo simulation can be used.32 The details of such models are beyond
the scope of this chapter, but it should be emphasized, first, that each model works accurately
only for a defined and restricted set of circumstances and second, that the optical properties must
generally be known to good accuracy — for example, in the scatter-dominated case, an error of
10% in µa and µ 's results in a 10% error in µeff and hence to a 30–40% error in the estimated
fluence rate at a depth of three penetrations depths and to correspondingly greater error at larger
depths. Thus, even if the general distribution of fluence rate is to be based on calculation, it is
helpful to use interstitial detector measurements at a few points as a check. Particularly in the
visible and near-IR, where there is considerable tissue-to-tissue and even subject-to-subject vari-
ation in absorption and scattering, such calculations of the fluence beg the question of how to
measure the optical properties. At other wavelengths, it is generally adequate to use generic values
for each tissue, often determined from measurements made ex vivo. For homogeneous tissues, the
absorption and scattering values can be determined either invasively or noninvasively, as illustrated
in Figure 3.19.
• Invasive measurements — The values of µa and µ's can be obtained by using two or more
isotropic detectors interstitially to measure the fluence rate, φ, at different known radial dis-
tances, r, from an isotropic source (or at different depths, d, during surface irradiation). For
example, if µ's >> µa, then diffusion theory can be used and the values of µa and µ's calculated18
using Equation 3.6b. It is an important aspect of this method that the absolute fluence must be
measured. Only dependent variables can be derived from a relative measurement such as φi/ φj:
φ (d1) φ (r1)
φ (d2) φ (r2)
d1
r1
d2 (a) r2
Tissue Tissue
R(ρ)
ρ1 ρ2 ρ
3
Tissue
(b)
FIGURE 3.19 Determination of tissue optical properties by (a) interstitial measurement of the local fluence at
multiple points with external or interstitial irradiation, (b) surface measurements of the local diffuse reflectance as
a function of the radial distance from the source on the tissue surface.
e.g., for an interstitial source and interstitial measurements, µeff = {ln(riφi/rjφj)}/(rj – ri) where
the subscripts refer to different radial positions in the diffusion region. The accuracy of these
calculations is increased if more than two measurement points are used, in which case, a best
fit to the diffusion equations can be performed.19
If only relative measurements can be made, yielding µeff, then, because the total diffuse
reflectance, Rd, is uniquely dependent on the transport albedo a' = µ's/(µa + µ's), also, measuring
this allows both µa and µ's to be determined (using Equation( 3.4)) from
µ a = µeff [(l – a')/3]1/2 (3.7a)
µ's = µeff ⋅ a'/[3(1-a')]–1/2 (3.7b)
If the tissue is not scattering enough or the homogenous volume is too small (e.g., in the
case of a solid tumor) to permit the use of diffusion theory, then multiple-point fluence
measurements (with or without measurement) still may permit the optical interaction param-
eters to be estimated, but more complex modeling is required. Note, however, that in this case,
it may be necessary to also determine the scattering anisotropy, g, because the fluence distri-
bution may not depend simply on µa and µ's. This requires techniques that have not yet come
into routine practice.
• Noninvasive measurements — As shown in Figure 3.19b, µa and µ's can be determined by
making multiple measurements of the radial dependence from a point source of the local diffuse
reflectance, R(ρ). Again, if the diffusion conditions apply, R(ρ) can be expressed uniquely in
terms of µa and µ's, so that the reflectance curve can be fitted to derive these coefficients,33 or
a neural network that has been trained on model or experimental data can be used.34 It has
been shown that absolute reflectance measurements are not required if the range of ρ values is
chosen appropriately (typically ρ/2<ρ<5ρ), because µa and µ's can be derived simply from the
t t t
t
t t
Tissue Tissue
(a) (b)
FIGURE 3.20 Time-of-f1ight measurements in tissue: (a) direct time domain, showing the temporal spreading (ns
time scale) of the signal following a short (ps) input pulse, (b) frequency domain, showing the phase shift and
demodulation of the detected signal with reference to the high frequency ( >~ 100 MHz) modulated input light.
The tissue absorption and (transport) scattering coefficients can be derived from the detected signal, knowing the
source-to-detector separation (which can be zero, allowing endoscopic or intravascular single fiber probes).
shape of the reflectance curve. Optical fiber-based instruments have been developed, the most
recent utilizing a broad-band source and spectrograph coupled to a 2-D CCD array to permit
measurements of R(ρ) simultaneously at each ρ value and for a range of wavelengths.35 This
has been used for noninvasive absorption spectroscopy in vivo, to measure µa(λ), from which
specific chromophore features can be identified and their tissue concentration estimated.
Recent modifications to the local diffuse reflectance method have been introduced, based on the photon
“time-of-flight” in tissue4,36 as illustrated in Figure 3.20a. The principle is that photons undergoing multiple
scattering between the source and a detector on the surface are delayed due to their variable pathlengths,
l, through the tissue: time delay = l/ c', where c' is the speed of light in tissue (=co/n, with co = 3 × 1010
cm.s–1 and n = tissue refractive index). Hence, if a short (~picosecond) pulse is incident on the tissue,
then the detected pulse is broadened by the variable time delay (different pathlengths), and the shape of
this time curve depends on µa and µ's (or also on µs and g at very short times). An equivalent method,
illustrated in Figure 3.20b, uses intensity-modulated incident light (typically at a frequency ~100–500
MHz) and measures the phase shift and demodulation of the detected signal with respect to the input.4,36
Again, µa and µ's can be determined. A significant advantage of these time-dependent measurements is
that the reflectance needs to be measured only at a single point on the tissue surface, and this can be close
to the input source. Thus, endoscopic techniques become feasible. Their principal disadvantage at present
is the technological complexity and cost compared with the steady-state, spatially resolved method. How-
ever, clinical systems based on light emitting diodes (LED) or diode laser sources are becoming available.
The application of these noninvasive methods for cases of heterogeneous tissues is not clear. The local
reflectance signals can certainly be affected by heterogeneity, as in the case, for example, of different tissue
layers.37 However, it is not known if this will be sensitive or specific enough to determine the distribution
of optical properties with the accuracy required for clinical fluence-rate calculations, for example, in skin.
Finally, for some cases of layered tissues, and possibly also for localized highly absorbing centers within
an otherwise homogenous volume, the technique of pulsed photothermal radiometry (PPTR) may play
a role, at least for wavelengths where the absorption is significant.38 As shown in Figure 3.21, infrared
radiation (black body) is emitted from the tissue surface as the heat generated within the tissue by the
absorption of light energy diffuses to the surface, following a short (~µs) input pulse at the wavelength
of interest. As the initial heat distribution depends on the distribution of the light fluence and of the
optical absorbers, the PPTR signal can be analyzed to estimate the optical absorption and scattering
Input Pulse
I.R. Signal
t(µs) I.R. Detector

Laser
t(ms)
I.R. Lens
Tissue
FIGURE 3.21 Illustration of the principle of pulsed photothermal radiometry, for the case where the incident light
pulse (~ µs) at the wavelength of interest is delivered to the tissue surface by an optical fiber and the emitted infrared
radiation is collected by a lens and focused onto an IR sensitive photo detector (e.g., HgCdTe photodiode). The tissue
optical properties at the irradiation wavelength are derived from the output signal magnitude and time dependence.
properties of the tissue itself39 or to measure absorbing exogenous dyes.40 It might also be possible to
determine some information on the spatial distribution of localized absorbers such as a blood layer.41
3.3 Thermal Dosimetry

In this section, the measurement of temperature distributions in tissue resulting from laser irradiation
will be considered.
3.3.1 Introduction
Many methods are available for measuring temperature changes in tissues resulting from the absorption
of laser energy. Most of the methods currently in use were developed originally for industrial applications
and then adapted to biomedical applications such as vascular imaging, tumor imaging and hyperthermia
monitoring.42,43
The principal forms of tissue damage employed in surgical laser applications are photocoagulation,
occurring at temperatures greater than approximately 60° C44 and vaporization at temperatures greater
than 100° C. Although both of these are thermal effects, thermal monitoring is rarely performed during
clinical laser surgery. In many established clinical applications, thermal tissue damage is well localized
and predictable, rendering unnecessary the acquisition of temperature information. This is fortunate,
because the task of measuring tissue temperatures with good spatial resolution and reasonable accuracy
is formidable in the large local thermal gradients found in laser surgery sites.
Thermal monitoring of laser-heated tissues has, in most cases, been restricted to laboratory investiga-
tions of effectiveness, optimization and safety that involve thermal dosimetry. Examples are temperature
measurements during laser angioplasty;45–51 laryngeal surgery;52 laser photocoagulation of the bladder
wall,53–56 abdominal wall,57 retina,58,59 brain,60–62 stomach,63,64 port-wine stain;65–68 and pulpal damage
during dental surgery.69,70
There is, however, a medical laser technique in which small solid tumors (smaller than approximately
10 cm3) are destroyed thermally by laser energy directed through implanted optical fibers, and for which
thermal monitoring may become an integral component of the treatment. This “minimally invasive” tech-
nique, known as interstitial laser photocoagulation (ILP) or interstitial thermotherapy (ITT), involves
delivering continuous laser energy at relatively low power (approximately 2 W per fiber) with a long exposure
duration (~1000s). ILP is currently being investigated for the destruction of primary tumors or isolated
metastases in the liver or pancreas,71–73 brain,74–76 and head and neck.77,78 Still lower delivered powers (less
than 1 W per fiber) can be used to induce hyperthermia, a technique referred to as interstitial laser
hyperthermia (ILH),79 possibly for use in combination with other modalities such as photodynamic therapy.
Four general methods of thermal monitoring can be identified: thermography, electrical probes, optical
fiber probes and radiologic imaging. As shown in Table 3.3, these methods can be distinguished according
to their invasiveness, portability and dimensionality (i.e., the number of spatial dimensions over which
temperature data can be collected).
TABLE 3.3 Practical Characteristics of Methods of Thermal Monitoring in Vivo

Method Invasive Portable Dimensionality
Electrical Probes yes yes 0 (point) or 1 (line)

Optical Fiber Probes yes yes 0 or 1
Thermography no yes 2 (surface)
Radiologic Imaging no no 2 (cross-sectional) or 3 (volume)
Generally, thermography and radiologic imaging methods offer the advantages of noninvasiveness and
multidimensionality, but at the expense of accuracy and spatial resolution. In biomedical laser applica-
tions, most temperature measurements are made using electrical probes or thermography, although
increasing use of optical fiber probes might be anticipated. Most of the following discussion will focus
on thermography and electrical temperature probes. Only brief summaries of thermal monitoring using
optical fiber probes and radiologic imaging will be given.
3.3.2 Thermography
Thermography is a noninvasive technique in which temperatures are monitored, recorded and dis-
played in a two-dimensional image, thereby allowing visualization of both thermal equilibrium and
transient heating patterns.42 There are three types of thermography: liquid crystal, infrared and micro-
wave thermography. Liquid crystal and infrared thermography are used to map surface temperatures,
whereas microwave thermography can map subcutaneous temperatures. Of these three, only infrared
thermography has been used appreciably in the biomedical laser field. The discussion here will focus,
therefore, on this technique, although brief discussions of liquid crystal and microwave thermography
will also be provided, since these could find biomedical laser application, for example, in monitoring
ILP or ILH.
3.3.2.1 Infrared Thermography — Basic Principles
Infrared thermography involves the detection, by an infrared camera (pyrometer), of the electromagnetic
radiation emitted from a surface at infrared wavelengths. The spectrum and total power emitted are
governed by the blackbody radiation laws: Planck’s Radiation law (spectral distribution), the Wien
Displacement law (spectral peak) and the Stefan-Boltzmann law (total emitted power).
A black body is an object that absorbs all radiation incident upon it, reflecting and transmitting none.
Most objects can be described as “gray bodies,” with radiative emission relative to a blackbody described
by the emissivity, ε (0 ≤ ε ≤ 1) and with total emitted power, E (W . m–2), at absolute temperature T
given by the modified Stefan-Boltzmann law:
E = εσT 4 (3.8)
Camera
Optical Path
Patient
Optics Field
Detector of
Video View
Display
Microprocessor
FIGURE 3.22 Principle of infrared thermography, showing temperature mapping by a scanned detector field of view.
where σ is the Stefan-Boltzmann constant, 5.672 10–8 W . m–2 . oK–4. Human skin, for example, is nearly
a blackbody at infrared wavelengths (ε ≈ 0.98).79
The spectral bands 3–5 µm and 8–12 µm are used for infrared thermography, because this minimizes
absorption in air. The thermal detector is usually a crystal of mercury-cadmium-telluride or, for 3–5µm
radiation, indium-antimonide,48 in which the electrical conductivity varies with the total radiant power
received, and which, in turn, is a function of surface temperature and emissivity. In practice, a measured
voltage is translated through a calibration table to gray level intensity or a color band for display on
a video monitor.48 (Conversion to a calibrated temperature map requires knowledge of the tissue
emissivity.)
An infrared camera is shown schematically in Figure 3.22. Temperature mapping is performed by
optically scanning the narrow field of view of the detector, using either rotating prisms or oscillating
mirrors. Commercial cameras typically have spatial resolutions of approximately 1 mm and temperature
resolutions of better than 0.2° C.
3.3.2.2 Infrared Thermography — Biomedical Laser Applications
In general medicine, infrared thermography has been applied primarily in the diagnosis of circulatory
disorders and vascular disease, breast cancer and joint inflammation.42,43
In the biomedical laser field,48 infrared thermography applications are essentially limited to research
studies. Typically, infrared cameras are used in experimental configurations to map tissue surface tem-
perature distributions per se, or for radiometric inflammation.42,43
Examples of surface temperature mapping are shown in Figure 3.23. The first illustrates measurement
of the heat accumulation and thermal damage following non-ablative CO2 1aser pulses.52 Note that the
CO21aser wavelength, 10.6 µm, falls within the detection band of cameras with mercury-cadmium-
telluride detectors, so that temperature measurements made during an irradiation may be erroneous,
due to detection of reflected radiation. Figure 23b illustrates a study in which a tissue phantom was used
to model retinal photocoagulation during argon laser irradiation.59
Both the safety and efficacy of laser photocoagulation can be assessed using infrared thermography,
for example, to estimate bladder wall temperatures during transurethral laser coagulation,54 to measure
skin surface temperature during argon or tunable dye laser photocoagulation of port-wine stains,68 or to
evaluate port-wine stain blood perfusion before and after argon laser therapy.65
Infrared cameras are also used to perform radiometry of laser wavelengths lying outside the two
infrared detection bands. This involves wavelength conversion through absorption of the laser wavelength
CO2 Laser Argon Laser
Infrared
Camera
Coagulated
Water Egg White
Handpiece
Plastic Wrap
Absorbing
Layer
Biological Material Infrared

Camera
(a) (b)
FIGURE 3.23 Examples of the application of infrared thermography: (a) to investigate heat accumulation and
thermal damage to tissue following non-ablative CO2 laser pulses,50 (b) to model retinal photocoagulation, measuring
the temperature changes induced in an absorbing layer.57
Black Body
Nd : YAG Laser
Beam (1064 nm) Sapphire Probe
Infrared
Camera
λ2
Darkened Surface
λ1
Glass Hemisphere
Tissue Sample Infrared

Camera
Heat
λ1= 1.064 µm Gun
λ2= 2 - 5.8 µm
(a) (b)
FIGURE 3.24 Radiometric applications of infrared cameras: (a) the use of a darkened external surface of a glass
hemisphere to convert Nd:YAG laser energy (1.064 pm) reflected from a tissue sample into thermal energy,61 (b)
measurement of the emissivity of a sapphire fiber tip probe.48
and re-emission of a thermal spectrum. Figure 3.24a illustrates a radiometric study in which the Nd:YAG
power transmitted and reflected from a tissue sample was converted on black-painted glass hemispheres
and then measured with an infrared camera.63
A further radiometric application of infrared cameras is the determination of surface emissivity. This
might be either an endpoint in itself or an intermediate step for temperature calibration. Emissivity can
be determined by aiming the camera on the object placed in thermal equilibrium with a heated black
body, and then adjusting the emissivity setting until there is no apparent temperature difference between
Transparent
Plastic Frame
Patient
Liquid Crystal
Material In
Inflated Balloon
Air Pump
FIGURE 3.25 Direct observation of thermal color map using liquid crystal thermography.
the tissue and the reference. Figure 3.24b illustrates one such experiment, to determine the emissivity of
sapphire probes mounted as optical fiber tips for use in laser angioplasty.50
3.3.2.3 Liquid Crystal Thermography
Liquid crystals utilized for thermography are organic compounds, such as cholesterol derivatives, that
selectively reflect visible light in a narrow, temperature-dependent range of wavelengths. When incor-
porated into a contact applicator, such as the inflatable balloon applicator depicted in Figure 3.25,
they can be used to generate color maps that enable direct observation of a cutaneous thermal pattern.
The color changes with progressive temperature elevation from longer to shorter wavelengths (reddish
brown to dark blue). Liquid crystal thermography systems are simple, portable, commercially available
and inexpensive. Biomedical applications include breast cancer detection81 and imaging of spinal root
compression syndromes.82 Medical laser applications of liquid crystal thermography are limited by the
contact nature of the technique.
3.3.2.4 Microwave Thermography
Microwave thermography involves the detection of electromagnetic radiation emitted from the body
at microwave frequencies (approximately 1–70 GHz). Unlike infrared and liquid crystal thermography,
this allows reception of the thermal signals arising from subsurface tissues. The maximum sampling
depth is determined by frequency. Microwave penetration in water decreases from approximately 3.3cm
at 2.45GHz (a commonly used frequency) to 0.3cm at 9GHz.43 Conversely, spatial resolution increases
with frequency.
In microwave thermography, the thermal signal (microwave noise) emitted from the body is measured
using a radiometer, consisting of an antenna and receiver. Both contact radiometers (1–10 GHz) and
offset, or “spaced,” radiometers with focused antennas (1-7OGHz) are used, as shown in Figure 3.26. In
the approximation that the probe–tissue coupling is optimal (black body), the power, P, collected by the
receiver from a tissue volume at uniform temperature T is
P = kT∆f (3.9)
where k = Boltzmann constant (1 . 38 10–23 J . °K–l) and ∆f = receiver bandwidth (Hz). In practice, suboptimal
coupling and tissue attenuation reduce the signal. Scanning is performed, as in infrared thermography, and
the microwave signal is digitized, processed and displayed as a color image of temperature distribution.
Biomedical applications of microwave thermography include cancer detection (breast, brain, thy-
roid) and noninvasive control of hyperthermia.83 Medical laser applications are likely to be confined
to temperature imaging of internal (interstitial or endoscopic) laser irradiation, and may be limited
by spatial resolution.
3.3.3 Electrical Probes

Unlike thermography, electrical probes for measuring tissue temperatures are implanted rather than
applied externally. The four most common electrical temperature transducers are thermocouples, ther-
mistors, resistive-temperature detectors (RTOs) and integrated circuit sensors. Practical descriptions of
the basic operating principles of these devices can be found in commercial temperature measurement
catalogs, such as Reference 84. RTOs are the most accurate and stable, but are expensive and seldom used
clinically. The discussion here will be confined to thermocouples and thermistors, which are the only
temperature probes commonly used in biomedical (laser) applications.
Offset
Receiver
1m
Reflector Patient
Video
Display
Contact
Receiver
Microprocessor
FIGURE 3.26 Microwave thermography using a contact or offset (focused ellipsoidal) receiver.
3.3.3.1 Thermocouples — Basic Principles

When two wires composed of dissimilar metals are joined at one end, a voltage is generated across the
open ends of the wires, as indicated in Figure 3.27a. This thermoelectric (“Seebeck”) voltage, V, is a
function of the sensing junction temperature and the composition of the two metals. For small changes
in temperature, V increases linearly with the sensing junction temperature, T, through the Seebeck
coefficient, α = dV/dT.
The most common bimetallic combinations are summarized in Table 3.4. Each of these has a nonlinear
temperature vs. voltage characteristic (see Figure 3.28) that is usually fitted by a high-order polynomial
over a wide temperature range or a low-order polynomial over a narrow temperature range.
At least one other junction in the measuring circuit must generate a thermoelectric voltage in series
with V. In practice, circuits can be constructed with one such junction maintained at a known reference
temperature, Tref, such as in an ice bath, and with all other junctions generating opposing voltages (see
Figure 3.27b). Alternatively, both voltmeter leads can be extended to reference junctions located on an
electrically insulating isothermal block, in which case, Tref is measured by a thermistor or RTO
(Figure 3.27c). In both cases, Tref corresponds to a reference junction voltage, Vref, that can be subtracted
from the measured voltage to give V.
Thermocouples are the most versatile but least sensitive of electrical temperature probes; a typical
thermocouple generates only 40 µV/° C. They are, however, rugged, inexpensive, usable over a wide
temperature range and simple, although requiring a reference temperature. Thermocouple temperature
data can be acquired using a voltmeter or a commercially available data acquisition system with capability
for multi-sensor input and computer interfacing.
3.3.3.2. Thermistors — Basic Principles
Increasing the temperature of a semiconductor decreases its resistivity by increasing the number of
electrons excited thermally from the valence to the conduction band. The resistance (R) vs. absolute
temperature (T) can be closely approximated by the Steinhart-Hart equation.84
TABLE 3.4 Common Thermocouple Metal Combinations

Type E J K R S T
+Metal Chromel Iron Chromel Platinum — Platinum — Copper

13% Rhodium 10% Rhodium
–Metal Constantan Constantan Alumel Platinum Platinum Constantan
Isothermal
Block
Voltmeter Voltmeter Voltmeter Cu
+ Cu + Cu A + Cu A
A J J J
Cu V Cu V Cu V
B
- - B - Cu
A B
Ice Bath R
Tref = 0oC
(a) (b) (c)

FIGURE 3.27 Principle of the thermocouple showing dissimilar metals, A and B, generating a thermoelectric voltage,
V, at their temperature sensing junction, J. At least one other junction in the voltage measuring circuit also consists
of dissimilar metals (a) and contributes a separate voltage determined using a reference junction such as an ice bath
(b) or isothermal block (c).
80
E
60
Voltage (mV)
40 J
20 R
T
S
0
500 1000 1500 2000
o
Temperature ( C)
FIGURE 3.28 Temperature vs. voltage characteristic for the six common thermocouple combinations listed in
Table 3.5.82
1/T = Cl + C2lnR + C3(lnR)3 (3.10)
where Cl, C2 and C3 are material-dependent constants. Thus, thermistors are highly nonlinear devices.
A typical R vs. T characteristic is shown in Figure 3.29.85 Linearized thermistors have been developed,
although the use of computerized data acquisition generally renders this unnecessary.
Thermistors are manufactured from oxides of nickel, manganese, iron, magnesium, copper, cobalt,
titanium and other metals. They are the most fragile but also the most sensitive of electrical temperature
probes, with temperature coefficients of resistivity typically around 4% per °C. A disadvantage is their
106
104
Resistivity (Ω.cm)
102
10-2
0 2 4 6 8
1000/Temperature (oK-1)
FIGURE 3.29 Resistivity vs. temperature characteristic of an Fe3O4 . MgCr2O4 thermistor.83
limited temperature range. The recommended maximum operating temperature is typically 100°C.
Higher temperature operation is possible, although at the risk of permanent loss of calibration.84
3.3.3.3 Thermocouples and Thermistors — Biomedical Laser Applications
Thermocouples and thermistors are useful for measuring temperatures in biomedical applications
because they can be constructed with very small dimensions. So-called microthermocouples consist
of two interwound wires with a combined outer diameter <<1 mm. These implantable temperature
probes are available commercially, either bare or with a Teflon coating to provide electrical insulation
and waterproofing. The smallest commercially available thermistors are also manufactured in tubular
geometry, with diameters as small as 1 mm. Another feature useful in biomedical applications is
that both devices have short response times, in the order of 1 s for thermistors and 0.1 s for
microthermocouples.
In the biomedical laser field, electrical temperature probes are most often used for real-time temper-
ature monitoring during interstitial laser heating of tumors (ILP and ILH), as illustrated in Figure 3.30.
Most of this work to date has been in animal models.79,86–90 Recently, ILP and ILH have been performed
clinically in the brain with stereotactic implantation of microthermocouples or thermistors to monitor
temperature,86,90 in the liver with implanted microthermocouples,73 and in head and neck cancer with a
single implanted microthermocouple.91
Electrical temperature probes have been used in surgical laser studies to measure temperatures in
normal tissues adjacent to the surgical target, for example, by implanting miniature thermistor probes
beyond the edge of a CO2 laser lesion “crater” to investigate tissue damage and healing in the treatment
of cervical intraepithelial neoplasia.57 Microthermocouples have also been applied to studies of the
thermal effects of Nd:YAG and CO2 laser irradiation using animal models.79,80,86–90 Recently, ILP has been
used clinically in brain with stereotactic implantation of electrical temperature probes.76
The safety and efficacy of laser angioplasty have been assessed using microthermocouples by measuring
the temperatures at or near the laser probe tip.49,51 The accuracy of these measurements may be limited
by possible conduction errors. Other studies have used K-type thermocouples attached to the outer walls
of harvested human femoral and popliteal arteries to help assess wall damage during laser angioplasty,51
as shown in Figure 3.31. A power meter for laser angioplasty has been developed based on temperature
increase in water measured using a miniature thermistor.45
With the increasing use of the CO2 laser for soft tissue surgery in dentistry there is greater potential
for thermal injury to teeth, which can result in irreversible pulpal damage. This problem has been
Optical Fiber
Stereotactic
Apparatus
Temperature Brain
Probe
Tumor
(a)
Optical Fibers
Temperature Probes
Ultrasound
Monitoring
Tumor
Liver
(b)
FIGURE 3.30 Interstitial laser photocoagulation (ILP) used to destroy inoperable tumors in (a) brain and (b) liver,
with temperature monitoring using implanted microthermocouples/thermistors. Computed Tomography or Mag-
netic Resonance Imaging can be used at the same time to monitor tissue response, while ultrasound can serve the
same function in the liver.70,73
Microthermocouples
Plaque
Laser Probe
Stenotic Artery
FIGURE 3.31 Microthermocouples attached to the outer wall and laser probe during laser angioplasty of a harvested
human stenotic artery (modified from Ref. 49).
investigated by implanting miniature thermistors in the pulp chamber of freshly extracted human molars
to assess thermal transfer to the pulp during laser irradiation of the external tooth surface.69
Electrical temperature probes can be made with intrinsic accuracy and precision of 0.1° C or better.
In practice, however, they are generally subject to three main types of error,92–95 two of which are
particularly relevant to laser applications. The first, “self-heating,” occurs when the probe directly absorbs
the laser energy more rapidly than the surrounding tissues, and so records too high a temperature. The
problem can be confounded by the probe, then heating the surrounding tissues. Self-heating errors arise
only when the temperature probe is located within approximately two effective optical penetration depths
(including geometric divergence) of the optical source, which is not usually the case.
The second error, “conduction,” is introduced by thermal conduction within the temperature probe,
resulting in poor spatial resolution. Conduction errors are greatest for metallic probes in high tem-
perature gradients. They often arise in biomedical laser measurements, but their effects have not been
well documented.
3.3.4 Optical Fiber Temperature Probes

Many concepts for fiber-optic temperature probes have been developed in the last two decades.96,97 Most
of these involve transmitting an optical input signal into one or more optical fibers, and then deducing
temperature from the optical signal returned by a sensor at the fiber tip. The sensors can be either
extrinsic, i.e., an added component located at the fiber tip, or intrinsic, in which sensing takes place in
the fiber itself (core, cladding or jacket). The three main types of optical fiber sensors are reflective,
fluorescent and interferometric.
Although commercial products are available, optical fiber sensors are more expensive than electrical
probes of comparable performance, and have not been widely used to date for biomedical temperature
measurements. Their principal advantage is that they are unaffected by electrical and magnetic fields and
so are well suited to applications such as temperature monitoring during magnetic resonance imaging,
or during heating from radiofrequency electromagnetic radiation or radiofrequency currents (e.g., micro-
wave hyperthermia).95
Because lasers have been used for distributed heating of small solid tumors71–79 and also in conjunction
with magnetic resonance imaging,75 it is possible that optical fibers could be used in the future for both
delivery of laser energy and temperature monitoring in the same application.
3.3.4.1 Optical Fiber Probes — Basic Principles
Five examples of reflective (extrinsic) optical fiber temperature sensors are illustrated in Figure 3.32.97
The first is a bimetallic reflector in which the differential thermal expansion of two metals in contact in
the sensor generates a displacement that alters the amount of incident light reflected. The second is also
a bimetallic sensor in which each metal forms a reflecting target, the relative displacement of which
creates temperature-dependent interference fringes. In Figure 3.32c, the intensity of reflected light is
determined by a crystal with temperature-dependent birefringence, while, in Figure 3.32d, the sensor is
a semiconductor crystal with a temperature-dependent absorption of infrared light. The sensor shown
in Figure 3.32e is a mirror that reflects light whose spectrum varies with temperature. The temperature
is then determined from the intensity ratio at two discrete wavelengths in the optical output signal.
In fluorescent sensors, incident ultraviolet light is absorbed and re-emitted at visible wavelengths.
Typically, the fluorescing element is a phosphor coating placed on the tip of a single encapsulated,
ultraviolet-transmitting fiber, as shown in Figure 3.33.98 Temperature measurement is based on the
temperature dependence of the intensity ratio at two discrete output wavelengths. Fluorescent sensors
are the most commonly used optical fiber temperature sensors in biomedical applications and have been
available commercially since the 1980s.
Interferometric temperature sensors are very sensitive to temperature-dependent changes in length
(as evidenced, for example, in Figure 3.32b) and refractive index.96,97 Interferometric probes require a
stable optical source, typically a gas laser. They are, therefore, relatively expensive and unlikely to be used
widely for biomedical applications in the near future.
Other optical fiber sensors have been developed in addition to the three main types discussed above.
Microbending of an optical fiber can be used to measure temperature;99 infrared transmitting optical
fibers can be used to detect infrared radiation emitted by heated tissues directly, avoiding completely the
requirement of an optical input source and an extrinsic sensing element; and infrared fiber radiometry
has been used to measure temperature transients following CO2 laser irradiation in tissue phantoms.100
3.3.5 Radiologic Imaging Methods

Certain radiologic imaging modalities can be used to measure temperatures in heated tissues. Unlike all
other methods of thermal monitoring, radiologic imaging potentially can provide full cross-sectional —
or even three-dimensional — temperature maps, although with poor spatial resolution compared with
discrete interstitial probes. Radiologic imaging methods are, therefore, better suited to temperature
Light source
Aluminum Mirror Silica
stalk substrate
Transmit leg Input
optical fiber
Thermal-contact From laser
plate
To
Single-mode monitoring
output optical fiber equipment
Receiving leg Bimetallic Silica Mirror
Detector element stalk
(a) BIMETALLIC DISPLACEMENT (b) BIMETALLIC INTERFERENCE
Protective jacket
Polarizer Mirror Sensing crystal
Transmit leg
LED Glass Fiber
tube
Detector
Receiving leg
Birefringent
crystal
(c) BIREFRINGENT CRYSTAL (d) SEMICONDUCTOR CRYSTAL
LED
Lens Connector
Sensor
Photodiodes Fiber
(e) SPECTRAL MODULATION
FIGURE 3.32 Various reflective fiber optic temperature probes. (Reprinted with permission from Fiber Optic Sensors,
2nd ed., Instrument Society of North America, 1992.)
Phosphor Layer Silica Core

(0.25mm thick) (0.4mm dia)
PFA Sheath
Silicone Cladding (0.7mm O.D.)
PFA PFA Encapsulation
(0.25mm thick) (0.8mm max. O.D.5mm long)
FIGURE 3.33 Fluorescent optical fiber temperature sensor. (Reprinted with permission from Biomedical Thermology.
Alan R. Liss, Inc. 1982.)
monitoring during cancer hyperthermia, which involves spatially distributed heating, than to measuring
localized temperature increases during laser irradiation.
Perhaps the most promising imaging modality for thermal monitoring is diffusion-sensitive magnetic
resonance (MR) imaging.101 This involves calculating the molecular diffusion coefficient, D, from a ratio
of signal intensities in two MR images differently sensitized to diffusion, and then calculating temperature,
T, based on the Stokes-Einstein relation
D ∝ exp(–Ea/(kT)) (3.11)
where Ea is the activation energy for translational molecular diffusion.

Thermometry based on ultrasound velocimetry has also been described.102 In this approach, a tem-
perature map is calculated from a map of ultrasound velocity, based on prior knowledge of the coefficient
of velocity variation with temperature.
3.4 Summary
Determination of light fluence distributions in tissue prior to or during clinical laser irradiation is becom-
ing practical due to the development of both invasive and noninvasive optical fiber-based techniques.
These techniques have resulted from improved understanding of tissue optical properties and light prop-
agation in tissue. As laser-based therapeutic techniques become more sophisticated, there is an increasing
reliance on accurate light dosimetry to ensure optimal therapy, and both general-purpose and application-
specific instruments are expected to become an integral part of medical laser systems in the future.
The principal forms of tissue damage exploited in many current clinical applications of surgical lasers
are thermally induced, and a wide variety of thermal monitoring tools are available. Yet, to date, such
monitoring of laser-irradiated tissues has been restricted primarily to pre-clinical laboratory investiga-
tions. A notable exception is interstitial photocoagulation of solid internal tumors, for which some form
of in situ treatment monitoring is usually considered essential. Given that thermal monitoring enables
important quantification of tissue damage during laser therapy, increased clinical utilization of the
techniques described above can be anticipated.
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4
Uses and Effects of
Ultraviolet Radiation on
Cells and Tissues
List of Abbreviations Used in this Chapter .................................. 85

4.1 Introduction to Ultraviolet Radiation ................................ 86
4.2. A Division of the Ultraviolet for Photobiological Studies...86
Vacuum UV (10 nm–190 nm) • UV-C (190 nm–290 nm) • UV-
B (290 nm–320 nm) • UV-A (320 nm–380 nm) • Terrestrial
Solar UV (290 nm–380 nm)
4.3 UV Sources ........................................................................... 88
4.4 Absorption of Ultraviolet .................................................... 90
Division Between Ionizing and Non-Ionizing UV • Molecular
Absorption • Absorption by Cells • Absorption by Tissue
4.5 Direct vs. Indirect Effects of UV......................................... 96
4.6 Action Spectroscopy: Effect as a Function of λ................. 96
UV-C (190 nm–290 nm) Action Spectra • UV-A (320 nm–380
nm) Action Spectra
4.7 Effects of UV on Cells ....................................................... 100
Cell Death • Cell Mutation • Membrane effects
4.8 Complex Responses of Tissues.......................................... 101
Immune Effects • Carcinogenesis
4.9 Repair .................................................................................. 103
4.10 General Effects.................................................................... 104
4.11 A Perfect Laser for UV Photobiological Studies.............. 104
Thomas P. Coohill 4.12 Useful Review Texts............................................................ 105
Siena College References ...................................................................................... 105
List of Abbreviations used in this Chapter

AS action spectra
eV electron volt = 1.6 × 10–19 J
λ wavelength(s)
UV λ 10 nm – 380 nm
UV-A λ 320 nm – 380 nm
UV-B λ 290 nm – 320 nm
UV-C λ 190 nm – 290 nm
VUV λ10 nm – 190 nm
0-8493-1146-2/02/$0.00+$1.50
4.1 Introduction to Ultraviolet Radiation

The known electromagnetic spectrum is open-ended and continuous, having no inherent upper and
lower bounds. It extends from the longest measured wavelength (about 104m) radio waves to the shortest
measured wavelength (about 10-16 nm) γ-rays.1 Of most interest to biologists — and hence life on earth
— is the terrestrial solar region (290 nm to longer than 2000 nm).1 This region contains those wave-
lengths emitted by the sun that penetrate the earth’s atmosphere to reach the surface, although techni-
cally any wavelength reaching the biosphere (maximum height about 6 km) can affect living processes.
Solar radiation is responsible for almost all of the energy added to the biosphere and drives such
important bioreactions as photosynthesis and vision. Studies of the effects of non-ionizing electromag-
netic radiation on living systems is known as photobiology. In addition, some photobiological research
is conducted using ultraviolet (UV) wavelengths that are shorter than those contained in terrestrial
solar radiation (i.e., shorter than 290 nm) This chapter limits itself to a discussion of the uses and effects
of UV on cells and tissues.
Ultraviolet radiation is that portion of the electromagnetic spectrum that extends from the lower
wavelength limit of human vision (usually defined as 380 nm or 400 nm) to wavelengths as short as
about 10 nm, where it overlaps the x-ray region. In the natural environment, the shortest wavelength of
sunlight that can be routinely measured at the earth’s surface is about 290 nm, largely due to the
absorption properties of ozone and other atmospheric gases. So, the only environmentally relevant UV
region is from 290 nm to 380 nm. However, artificial UV sources such as certain fluorescent lamps,
mercury and xenon arcs, and lasers, are readily available and extend the possibility of human exposure
to UV down to wavelength limits of about 190 nm. Below 190 nm, air (oxygen) and water begin to
absorb UV heavily making it difficult2 to expose biological samples except under extreme conditions
(e.g., in a vacuum).
4.2. A Division of the Ultraviolet for Photobiological Studies

It is always difficult, and for some, unreasonable, to fractionate a continuum into convenient usable
regions. For example, red is defined as “very approximately,” the wavelength region 630 nm to 700 nm.3
Such imprecise definitions suffice for normal conversation, but more rigorous guidelines should prevail
for scientific presentation. This is especially important when the inclusion (or exclusion) of certain
wavelengths can markedly alter the biological response of the effect being studied (as is often the case
for the various definitions of the UV-B).
The naming of some of the regions of the electromagnetic spectrum is anthropocentric. The “visible”
region (380 nm–780 nm) defines the approximate region of normal human vision and excludes, for
example, cat vision in the infrared and insect vision in the UV. In fact, the term ultraviolet is itself vague
and replaced a hodgepodge of earlier terms such as “abiotic rays,” “chemical rays,” and “actinic rays.”4
The wavelength bounds for the UV region are as approximate as those for any other λ region, but the
term has proven useful. In a similar manner, the driving force for fractionating the biologically active
UV was the desire of those working with human skin to bracket regions based on erythemal (sunburn)
response. Although useful to dermatologists, these regions — defined as (UV-A, UV-B, and UV-C) were
for many years ignored by photobiologists. Thus, authors routinely used the terms near UV (near the
visible) and far UV (far from the visible), or various other definitions, to attempt to identify the region
of UV they were utilizing.
The conventions listed in Table 4.1 are not unique to the author. Rather, they are common in the
photobiological literature. They begin at the short wavelength (λ) end of the UV.
1 In some cases, the photon is defined by the method of production, not merely its λ, e.g., γ-rays and x-rays.
Uses and Effects of Ultraviolet Radiation on Cells and Tissues 87
TABLE 4.1 Common Fractionation of the Ultraviolet Region

Name λ Bounds in nm Comments — all approximate
Terrestrial Solar UV 290–380 Human vision occurs above 380 nm. 290 nm is shortest solar λ
measurable at sea level.
UV-A 320–380 Ozone relativity transparent above 320 nm.
UV-B 290–320 Dominated by ozone absorption of solar UV.
UV-C 190–290 Oxygen (air) and water transparent above 190 nm. These λ are
absent in terrestrial solar UV.
Vacuum UV 10–190 Mostly ionizing photons. Absorbed heavily by water and air.
Note: The definitions of the terms UV-A, UV-B, and UV-C vary with author. Those given above are commonly
used by photobiologists. Other definitions vary by no more than about 10 nm for any given λ bound.
See text for details.
4.2.1 Vacuum UV (10 nm–190 nm)

There is no general agreement as to where the UV ends and x-rays begin. Most textbooks show the two
regions overlapping. Hence, authors have cited λ as short as 1 nm as UV. Some photobiologists limit the
UV to λ above 100 nm. A reasonable compromise is 10 nm. Photons in the vacuum UV (VUV) are
heavily absorbed by water and oxygen (in air), both of which become essentially transparent (more than
50% transmission for a one cm path length)5 to UV at λ above 190 nm. Because of this limited penetration,
VUV damage to cells is usually confined to a narrow region (a few microns) near the cell surface. The
energies of single photons in this region are above 6.5 eV, sufficient to ionize many biomolecules.6 Because
the biological effects due to ionizing photons are different from those due to non-ionizing photons, the
vacuum UV often causes different types of cellular and molecular damage. Thus, vacuum UV effects can
be qualitatively different from those of other UV regions. Frequently it is convenient to generally regard
λ below 190 nm as ionizing and above 190 nm as non-ionizing, although the energy needed to reach
the ionization band of molecules varies widely (see below).
4.2.2 UV-C (190 nm–290 nm)

The shorter λ end of the UV-C (190 nm) is the λ region, where air and water become transparent. The
longer λ limit (290 nm) is the shortest solar UV λ easily measured at the earth’s surface. Thus, by this
convention, all of the UV-C is environmentally irrelevant. However, research in the UV-C range was
central in elucidating many important features of cell functioning. DNA, the genetic material, has a peak
absorption near 260 nm, which falls by a factor of six by 290 nm. This fact, combined with the readily
available 254 nm “germicidal” UV fluorescent source, allowed for simple experimental molecular manip-
ulation of DNA, and UV-C photobiology was central in establishing the then-new field of molecular
biology. Accordingly, a considerable body of work that unraveled cellular function was accomplished in
this environmentally irrelevant region.7
4.2.3 UV-B (290 nm–320 nm)

The short λ limit (290 nm) of the UV-B can be defined as the shortest UV λ routinely measurable at the
earth’s surface, where it is about one million-fold less prevalent than 320 nm radiation. The long λ limit
(320 nm) is where ozone and other atmospheric components begin to appreciably (more than 50%)
attenuate sunlight. This absorption prevents much of the significant DNA damage that would result if
no ozone layer existed. It is thought by some that the limited absorption of DNA in the UV-B (compared
with the UV-C) is, in part, due to the evolution of life under the protective ozone screen. DNA absorption
rapidly decreases toward the longer λ end of this region.8 Nevertheless, absorption of UV-B photons by
DNA contributes to a wide variety of bioeffects. Stratospheric ozone depletion will cause an increase in
the amount of UV reaching the biosphere and this increase will be fully contained within the UV-B. The
UV-B is a transition region from the heavily damaging UV-C to the less damaging UV-A. The product
of solar UV exposure, and, for example, DNA absorption, peaks at about 300 nm.9 Therefore, the UV-
B is responsible for most of the damage inflicted on organisms by sunlight. This UV-B definition also
closely brackets the effective solar UV absorption properties of the ozone layer.
4.2.4 UV-A (320 nm–380 nm)

The shorter λ limit of the UV-A (320 nm) is defined here as that λ at which ozone becomes transparent.
The longer wavelength limit (380 nm) is where human vision begins and the UV ends. The UV-A is also
biologically effective and causes cellular death, mutation, and DNA damage. However, the primary
chromophores for these effects may be non-DNA sensitizers that are chemically matched to the photon
energy and act as intermediates in relaying the absorbed energy to DNA.10 It is also the region where
some photoreactivation can occur and is heavily absorbed by certain biopigments. Thus, the UV-A, which
is still not clearly understood, is proving to be a very complex area of photobiology. Some UV-A effects,
such as cellular mutation (which should ultimately be caused by effects in DNA) occur at UV-A exposures
far less than those thought to affect DNA. The absorption spectrum of DNA in the UV-A, though low,
is not clearly defined.8 Some UV-A effects seem to mimic those due to ionizing radiation although UV-
A photons do not ionize molecules. UV-A exposures from sunlight are considerable, constituting about
8% of the total sunlight spectrum (compared with less that 0.3% for the UV-B).
4.2.5 Terrestrial Solar UV (290 nm–380 nm)

Terrestrial solar UV is easily defined as the λ bracket that is the limit of human vision (380 nm) at the
long λ end, and the effective limit of the solar UV reaching the earth’s surface at the short λ (290 nm)
end. This is the environmentally relevant UV region and is, of course, as defined here, just the UV-B
plus the UV-A.
No clear fractionation of the biologically effective UV is ever as precise, or as desirable, as a detailed
description of the UV source, its output (both in λ distribution and intensity), and its measurement,
which should be clearly stated by experimenters. For example, there are no pure UV-B sources (unless
one considers monochromatic sources), so it is meaningless to describe a fluence of “10 jm–2 of UV-B.”
But, although the term UV-B cannot replace a detailed description, it is useful in defining the region of
UV being utilized.
4.3 UV Sources
A variety of UV sources exist for use in biomedical applications. As shown in Table 4.2, the selection of
an appropriate source for UV photobiological studies is often determined by availability and cost. Simple
polychromatic sources are useful for studying bioeffects; monochromators for action spectroscopy; and
synchrotrons and lasers for more sophisticated molecular effects and time-resolved spectroscopy. Poly-
chromatic sources include the sun, solar simulators, and fluorescent lamps.
TABLE 4.2 Comparison of UV Sources

Spectral
Source Purity Intensity Cost
Germicidal lamp High Medium Low

Polychromatic fluorescent — Medium Low
Solar simulator — Medium Medium
Monochromator Medium Medium Medium
Synchrontron High Medium* Highest
Laser Highest Highest High
* Medium intensity in the λ range above 190 nm; relatively high below
190 nm.
The advantage of solar radiation is that it is a normal component of the ambient radiation beneath
which humans evolved. The curative effects of solar exposure are ancient in use and include the taking
of plant (Ammi majus) extracts followed by solar exposure for the treatment of vitiligo. Detrimental
effects of solar UV will be discussed in a following section. However, the sun is an awkward source for
use in laboratory or therapeutic regimes. Its intensity and wavelength distribution vary in relation to
position on the earth’s surface, time of day, season, atmospheric conditions, etc. Of more direct use is a
solar simulator that filters the output from a xenon arc so that it more closely matches the terrestrial
solar output. It is often used in biomedical research.
The simplest polychromatic sources are fluorescent bulbs. They contain Hg gas atoms which, when
ionized by accelerated electrons, in turn excite other Hg atoms that emit photons according to the energy
level differences of the Hg atom, especially the strong resonance output at 254 nm. A special phosphor
coating on the inside of the bulb envelope converts these UV-C photons to UV-B, UV-A, and visible
radiation. Some bulbs are specific for each of these regions. The glass envelope attenuates most of the UV-C.
Certain fluorescent “sunlight” bulbs are also manufactured, but a careful check of their spectral
output is necessary to determine their properties. Even small amounts of short λ UV, less than 300
nm, can markedly affect the biological effects of these bulbs. Mylar filtration is advised. Incandescent
halogen lamps produce large amounts of UV. Monochromatic sources also vary in intensity, spectral
purity, wavelength range, cost, and ease of operation. A simple germicidal lamp acts the same as a
fluorescent bulb. It has no phosphor, but has a UV transparent envelope. It emits 86% of its radiation
at 254 nm.5 It fits into simple fluorescent sockets, such as a desk lamp, and is used in sterilization. The
nearness of the 254 nm to the peak absorption of DNA (260 nm) makes this an ideal source for many
photobiological experiments.
More-intense sources can be fractionated into monochromatic regions, each with sufficient intensity
to cause a biological effect. Mercury and xenon arcs are commonly used, often with the Hg-Xe mixed in
combination, and have proven useful for the wavelength region 220 nm–380 nm. Synchrotrons can
produce high intensity UV radiation that is also tunable, but one has to travel to the few available sites
to use them. But, for the widest potential application, nothing compares with the laser. Its spectral purity,
collimation, power, and reasonable availability make it an ideal source for many uses. If only a single
wavelength is needed (e.g., in matching the incident radiation to the peak absorption of a molecule) then
a simple laser is sufficient. If a variety of λ are required, a tunable laser is necessary. The limited availability
of inexpensive, tunable sources in the UV-C and UV-B has hampered the widespread use of lasers in UV
photobiology. Some action spectra (see below) have already have been reported using tunable lasers.
Recent advances in the area of UV and VUV lasers are covered in this book.
It is especially important that one choose the correct UV source for the needed purpose, is aware of
the absorbing and scattering effects of the materials (including gases and solutions) between the source
and the sample, and measures the intensity and λ distribution as close to the target sample as possible.
Table 4.3 lists the λ at which 50% and 90% of an incident beam of UV will be absorbed by some common
TABLE 4.3 Ultraviolet Absorption by Common Materials

λ for absorption of
Thickness 50% 90%
Ozone (0 °C) 1 cm 330 314

Air (O2 at 0 °C) 1 cm 181 174
Water 1 cm 189 186
Quartz (UV fused) 1 mm 164 160
Window glass 1 cm 340 332
Pyrex glass 1 cm 325 306
Note: There are several different types of quartz and glass; some aqueous
buffers will have higher UV absorption due to dissolved compo-
nents. Data from a variety of sources, especially Jagger, 1967.
TABLE 4.4 Approximate Photon Flux (Photons cm-2 sec-1) for Various Radiation Sources*
Source 100 nm 200 nm 300 nm 400 nm
Double monochromator — 2 × 10 13 2 × 10 15 1 × 1015

Tunable dye laser — - 2 × 1016 1.5 × 1016
Synchrotron 4 × 1014 4 × 1014 4 × 1014 2 × 1014
RF-Linac Free electron laser 7 × 1018 3 × 1019 5 × 1019 1 × 1020
Germicidal lamp — 1015 at 254 nm — —
Polychromatic fluorescent — 2 × 1014 from —
300–400nm
Solar simulator — — 2 × 1016 from —
290–400nm
Sunlight (noon) — — 1016 from —
290–400nm
* Sources vary greatly in bandwidth and spectral purity. For example, the spectral band width for the synchrotron
is 1%, for both lasers less than 0.1%, and for the monochromator about 2%. Wattage varies also.
These values are for a beam passed through a monochromator. A storage ring undulator may increase the
synchrotron readings by two orders of magnitude.
substances. Note the protective absorption of ozone beginning at about 330nm, which shields life forms
from detrimental UV exposure. Air and water do not absorb heavily at λ above about 190 nm. Quartz
is useful for photobiological studies because it transmits well at λ above 170 nm. Window glass begins
to absorb appreciably below 340 nm. All of these values change with thickness. No listings are given for
various plastics, which can have different absorption properties, even from batch to batch. In addition,
UV ages plastic rapidly, changing its absorption properties. It is especially important that one be aware
of everything in the path between sample and UV source. It is also crucial that the source be carefully
described in any publication as to type, output (Table 4.4), measured parameters, and experimental
configuration. If possible, each laboratory should carefully measure the output of any source using a
calibrated spectroradiometer.
4.4 Absorption of Ultraviolet

To a first approximation, the absorption of non-ionizing radiation follows the Beer-Lambert law:
I – nsx
---- = e
Io
where Io = incident intensity, I = final (sometimes transmitted) intensity, x = absorber thickness, n =

number of absorbers, and s = absorption cross section. Thus, absorption is, to the first order, an
exponential process. Hence, we also define:
I
log ----o = optical density or absorbance
I
The first law of photochemistry states that a photon must be absorbed to produce an effect. The
quantum yield (φ) is then defined as:
number of molecules photochemically altered

φ = --------------------------------------------------------------------------------------------------------------
number of molecules that absorb a photon
The range of φ in photochemistry is usually from about 10–6 up to the maximum value of 1.5
4.4.1 Division Between Ionizing and Non-Ionizing UV

There is an important division in the UV for radiation effects. In general, photons of wavelength shorter
than about 190 nm have enough energy to ionize atoms and molecules. But the ionization potential of
atoms and molecules is too high for wavelengths longer than 190 nm to have this effect. This distinction
between ionizing and non-ionizing UV is crucial, because the mechanisms of damage by each type of
radiation are different. Ionizing radiation cuts an energy deposition path through cells and tissues that
can cause a variety of indirect as well as direct effects, often by the formation of free radicals. Non-
ionizing radiation has to be absorbed to effect a change. Hence, a non-ionizing UV photon can traverse
a cell, not be absorbed, and the cell will have no history of exposure to it. Because almost all biomedical
uses of UV employ sources at wavelengths greater than 190 nm, only the non-ionizing portion of the
UV need concern us here. This is not to imply that ionizing UV research (in the vacuum UV) is
unimportant, or will not have biomedical applications in the future.2
4.4.2 Molecular Absorption

As mentioned above, the first rule of photochemistry is that a photon has to be absorbed to produce
an effect. Molecules absorb non-ionizing radiation by three fundamental mechanisms. As shown in
Figure 4.1, electrons, normally in the ground electronic state, can absorb photons directly and go to an
excited state that has an energy difference equal to the energy of the absorbed photon (E1 in Figure 4.1).
In addition, there are energy levels contained within the electronic levels that allow for changes in the
vibrational and rotational modes of molecules. Table 4.5 shows the energy requirements for photons to
excite the various energy levels present in molecules. In general, electrons can be excited into the
ionization band by λ in the VUV. The remaining UV and visible regions can excite electrons from the
ground state to various electronic excited states. Infrared radiation can excite molecules into higher
FIGURE 4.1 An energy level diagram for a diatomic molecule. Solid curved lines are the envelopes for the electronic
ground state and the first excited electronic state. Solid horizontal lines represent vibrational energy levels superim-
posed on the electronic ones. Not shown are rotational energy levels that would be superimposed on the vibrational
energy levels. Vertical lines; solid line represents the absorption by the molecule of a photon of energy E1 , dashed
lines the emission of a photon by the molecule, wavy lines the internal loss of energy by collision. For details see text.
TABLE 4.5 Absorption Region for Excitation of Biomolecules

Photon Energy to Approximate Spectral
Energy Level Excite in eV Region
Ionization band ≥6 Vacuum UV

Electronic 1.0 UV-A-B-C, visible
Vibrational 0.1 Infrared
Rotational 0.01 Microwave
Note: Real values depend on the molecular structure and vary accord-
ingly. 1eV = 1.6 × 10–19 J.
vibrational states; microwave radiation excites molecular rotational energy levels. As stated, these values
are rough approximations, as the structure of molecules determines their individual absorption proper-
ties. Figure 4.1 shows a typical energy level diagram for molecular absorption. As shown, an incident
photon of energy El, is absorbed by the molecule, raising a ground state electron to the first excited state.
Here it has a variety of ways to return to the ground state. It can do so directly by emitting a photon of
energy equal to E1 ; it can lose some energy of vibration by collision with other molecules and then emit
a lower energy photon E2; or it can emit an even lower energy photon E3; returning to a higher vibrational
level in the ground state, eventually going to the lowest vibrational energy level by collision. The last two
mechanisms mean that a photon of longer λ than was absorbed is emitted. In general, the length of the
conjugated double bond region of a molecule determines the long λ limit of absorption.
4.4.3 Absorption by Cells

Beginning at the cellular level, we can see from Table 4.6 that 200 nm UV-C is absorbed only slightly by
viruses (about 30%), more so by bacterial cells (about 70%), and entirely by mammalian cells (more
than 99%). This absorption is mainly due to the presence of endogenous molecules that absorb heavily
in the UV-C. Figure 4.2 compares the absorption of two important biomolecules, DNA, the genetic
material, and protein. Both molecules begin to absorb appreciably in the UV-B and substantially in the
UV-C. On a weight-by-weight comparison, DNA absorbs about 20-fold as much 260 nm radiation as
does protein. Therefore, the absorption of even homogenates of mammalian cells (Figure 4.3) is similar
in the UV-C, in wavelength dependence, to that of DNA. The absorbance (a logarithmic function) of
such homogenates is seven times higher at 240 nm than at 300 nm.11 As the UV wavelength decreases
below 220 nm, the large number of peptide bonds present in proteins begin to contribute greatly to the
total cellular absorption. At wavelengths below 190 nm, water and oxygen absorption prevails. In addition
to these macromolecules, some cells contain certain pigments that can alter cellular absorption signifi-
cantly, even at the longest UV wavelengths (Figure 4.4). Also, cellular particles and organelles can absorb
TABLE 4.6 Estimate of the Percent Transmission to the Center of Selected Cells and Viruses in the Ultraviolet
Wavelength in nm
Biological sample Diameter (µ) 200 250 300 350
Bacteriophage (T2) 0.1 74 86 100 100

Herpes simplex virus 0.15 66 80 100 100
Bacterial cell 1 33 78 98 100
Yeast cell 5 1.6 69 97 100
Mammalian cell (umbonate) 4.6 10–2 50 95 99
Mammalian cell (spherical) 20 10–7 20 91 96
Mammalian tissue (100 µ thick) — — 10–5 39 66
Note: All values are approximate; values at λ above 300 nm can vary widely due to the presence of endogenous chro-
mophores; umbonate is the flattened geometry of mammalian cells when attached to surfaces. See Coohill (1986)
and Coohill and Sutherland (1989) for more complete discussion.
FIGURE 4.2 The ultraviolet absorption of DNA and a typical protein. At short λ, the absorption of the numerous
peptide bonds in the protein predominate.
FIGURE 4.3 Absorption spectra for a homogenate of monkey kidney cells (CV-1) and human HeLa cells.
FIGURE 4.4 Absorption spectra for four important molecules in human skin. Data is averaged from several authors,
including Parrish et al.12 and Everett.48
and scatter UV radiation and, at some wavelengths, shield the center of the cell from a significant portion
of the incident beam. Such cytoplasmic screening can alter measured bioeffects to a large degree and
must be considered when cellular exposure is attempted.11 Due to the nature of living material, it is often
difficult, or impossible, to place the UV dosimeter at the site selected to be irradiated (e.g., the nucleus
of a cell). In such cases, it is important to measure the intensity of the incident beam at the surface of
the sample and on the exit side of the sample. Only then can a reasonable estimate of the exposure of
the target area to the beam be derived. In the case of living tissue, it is often impossible to place a dosimeter
on the exit side, so estimates must be made from separate measurements of the optical properties of the
tissue being exposed. Such concerns are important because biological cells and tissues will absorb
radiation in a wavelength-dependent manner and alter the exposure of the target accordingly. Figure 4.5
shows the penetration depth of various UV-C λ into mammalian cells. The horizontal lines show the
level at which approximately 50% of a beam at that λ will be absorbed. Cells in a spherical configuration
(e.g., in solution) absorb more heavily because the beam has to traverse more protoplasm. Cells in the
FIGURE 4.5 The approximate penetration depth into a single mammalian cell for 50% absorption of an ultraviolet
beam at different λ. Spherical cell (often the shape in solution), and flattened umbonate cell (often the shape in
tissue or cell culture). For example, 50% of a beam of 280 nm radiation makes it to the center of a spherical cell,
while more than 50% at that λ traverses an umbonate cell.
flattened (umbonate) configuration (e.g., in some tissues or in cell culture) allow more than 50% of a
beam of 280 nm to completely traverse the cell.
The absorption properties of cells are not merely a summation of the individual absorption of their
component molecules. Scattering of incident radiation by cellular structures (granules, mitochondria,
membranes, etc.) and the shielding of possible target chromophores by pigments and other molecules
and structures add to the complexity of predicting the absorption properties of any cell. However,
reasonable estimates can be made based largely on experimental measurement of cellular absorption
(when available) and the known absorption properties of cellular constituents.
4.4.4 Absorption by Tissue

It is surprising how many people think that tissue (e.g., skin) is opaque to light. Any children who
have gone into a dark closet, placed a lit flashlight into their mouths, and looked into a mirror, have
seen the red end of the visible spectrum emerging from their cheeks. Of more concern is the statement
by some scientists that UV does not penetrate into the human body. Of course UV, in a very wavelength-
dependent manner, does penetrate into certain organs such as the skin and eye. This penetration is
limited but, in turn, limits the uses of UV sources for biomedical purposes. In fact, tissue optics is a
reasonably well developed field, and long wavelength UV can penetrate at least 1 mm into the skin,
i.e., traversing the stratum corneum and epidermis, and going well into the dermis. Although this
penetration is highly dependent on skin type and pigmentation, about 40% of UV-A reaches the dermis
in white skin, and about 10% in black skin.12 This means that a significant amount of UV-A reaches
blood vessels where it can be absorbed by blood and other moieties. Cells circulating in the blood
system can also be affected.12
Much of the direct absorption of UV by the skin can be accounted for by the presence of endogenous
pigments, especially melanin, hemoglobin, carotenes, and keratin (Figure 4.4), and exogenous pigments
and drugs. Because skin is such a heterogeneous material, UV can be reflected, absorbed, scattered, and
re-scattered. This means that the direct component of the UV beam is augmented by additions from
photons scattered and reflected back into the beam pathway. Hence, at any tissue depth, the sum of the
total UV exposure is just the direct plus the diffuse. Table 4.7 is a very approximate estimate of the
percentage of UV at various wavelengths that will traverse the human epidermis. These values will vary
widely among various skin types, and can change as an individual adapts to UV exposure. Dry skin will
allow more penetration than wet, because water will reflect a potion of the incident beam away from the
skin. As shown, penetration of greater than 10% of the incident beam occurs for λ above 300 nm. Below
300 nm, absorption rapidly increases, making the skin essentially opaque to λ below about 290 nm. An
excellent series of charts showing the penetration of different λ of UV into human skin can be found in
Parrish, pp. 74–75.12
TABLE 4.7 Rough Estimates of the Percent of UV

Transmitted through Intact Human Epidermis
λ % Transmission
380 58
350 41
325 36
300 11
290 2
275 0.1
270–240 <1.0
Note: Can be highly variable; data for “normal” Cau-

casian skin; averaged from a variety of sources,
expecially Anderson and Parrish (1981) and Ever-
ett et al. (1966).
4.5 Direct vs. Indirect Effects of UV

Direct effects of UV exposure imply that the chromophore absorbing the radiation is changed so as to
cause a bioresponse. This is often the case for UV exposure at λ between 190 nm and about 320 nm.
Here, the effects of photon absorption by important molecules like DNA are largely known and include
such photochemical changes as the formation of pyrimidine dimers (Figure 4.6), 6–4 photoproducts,
DNA-protein cross links, and lesions that can lead to single and double strand breaks.13–15 If left unre-
paired, these lesions may lead to impairment, mutation, and even cell death. The biochemical and
physiological consequences of UV exposure of some biological systems are partially characterized and,
to some extent, understood16,17 when compared with other insults, e.g. ionizing radiation.18 However, in
the UV-A, it is not uncommon to observe events such as sensitization due to photodynamic effects
involving reactive oxygen species such as singlet oxygen and superoxide anion. There is also evidence for
the generation of H2O2 by UV-A and a consequent production of OH radicals. In some ways, these UV-
A effects mimic the results of exposure to ionizing radiation.
FIGURE 4.6 One example of a common cellular photoproduct after UV radiation, the pyrimidine, here thymine,
dimer. These dimers are formed by UV, and can be reversed by shorter λ UV, longer λ UV in the presence of the
photoreactivation enzyme (PHR), or by excision repair (ER) in the absence of light. See text for details.
4.6 Action Spectroscopy: Effect as a Function of λ
4.6.1 UV-C (190 nm — 290 nm) Action Spectra

Because some cellular molecules absorb heavily in the UV, certain bioresponses are highly dependent on
the λ to which the cell is exposed. Indeed, it is sometimes possible to determine the type of molecule
responsible for a given effect by comparing the absorption spectrum of the molecule to a spectrum
derived by measuring the cellular response as a function of λ. The latter is called an action spectrum
(AS), and was the first method of analysis to point to chlorophyll as the major chromophore in photo-
synthesis. Action spectroscopy has played a central role in the elucidation of the mechanisms of cellular
responses to UV radiation, especially the UV-C,5 and, as such, has contributed to our understanding of
the molecular biology of cells. In 1930, Gates (Figure 4.7) reported19 that the AS for bacterial cell death
closely followed the absorption of nucleic acid, not protein, which was then widely believed to be the
genetic material. In retrospect, this was the first clear evidence that DNA was the genetic material.
Although AS utilizing small cells is somewhat simple, it is difficult to extend these studies to larger (e.g.,
mammalian cells) because of the substantial absorption of UV by large cells and tissues (Table 4.6).
Figure 4.7 also shows two other AS in the UV-C for cell death, one for mammalian tissue20 and one
for individual mammalian cells.21 Early attempts by Mayer and Schreiber (1934)20 failed to produce AS
with the fine structure of those of Gates (1930)19 because they employed hanging-drop mammalian tissue
samples that were essentially opaque to radiation near the peak of DNA absorption (260 nm). The target
molecule (DNA) was shielded to an extent such that the shape of the AS did not parallel the absorption
FIGURE 4.7 The absorption spectrum for DNA, and action spectra for: bacterial cell death,19 cultured mammalian
cell death,21 and cell death in mammalian tissue.20 See text for details.
spectrum of the target. The advent of single cell mammalian culture techniques and the unique flattened
geometry that mammalian cells assume when in monolayer culture,11 allowed these studies to begin.
Thus, AS for killing of cultured mammalian cells21 reported data similar to those of Gates19 with bacteria,
but the peak was shifted to about 270 nm. This discrepancy can be accounted for if one looks at the
absorption properties of single mammalian cells and considers that, as is the case in bacteria, the likely
target molecule for cell killing is also DNA, which resides in the nucleus. This means that the UV beam
has to traverse, on average, half of the cell to strike its target. In bacteria, this distance is small enough
to allow one to neglect absorption effects; in mammalian cells the absorption is substantial and λ
dependent (Figure 4.3). However, because the target and primary photoproduct for this effect was already
known from work with bacteria, an AS for the production of pyrimidine dimers caused by UV exposure
of that target was measured. Thus, the effects of cytoplasmic shielding were accounted for in the exper-
imental results themselves. Accordingly, measurements of the AS for pyrimidine dimer production
matched the action spectrum for cell death in mammalian cells (Figure 4.8). Therefore, DNA alone was
responsible for mammalian cell lethality by UV-C.22
4.6.2. UV-A (320 nm — 380 nm) Action Spectra

It was widely thought that experimental work in the UV-C and UV-B (λ range 190 nm–320 nm) could be
extrapolated to predict photobiological responses in the UV-A (320 nm–380 nm). This is not the case, and
UV-A ASs are much more complex than had been predicted.10,17,23 Here, even events such as cell mutation,
which surely involves the genetic material, can be affected at fluences that appear to be below those necessary
to affect DNA in these λ regions. Figure 4.9 details the divergence of several ASs for human cell photore-
sponses from the DNA absorption spectrum as the curves shift to longer λ in the UV-A. In the UV-C, all
the measured effects, cell killing, mutagenesis, DNA breaks and cross-links, follow the absorption
FIGURE 4.8 The absorption spectrum for DNA and the averaged survival action spectrum for a large variety of
mammalian cell lines. The points are the wavelength dependence of pyrimidine dimer formation by exposure of
whole cells to UV. Note that these points closely follow the cell survival curve.7
spectrum of DNA. In the UV-B, breaks and cross-links are made at a rate above one that can be explained
by DNA absorption alone. In the UV-A, all of the biological parameters are affected at levels that are not
fully explainable by the absorption properties of DNA. The absolute absorption of moieties in the DNA
molecule itself is difficult to measure at λ above 320 nm because of scattering and contamination by
extraneous chromophores.8 It is thought that, if the absorption is not intrinsic to DNA, then an intermediate
molecule may be involved that absorbs the incident UV-A photon and transfers the effect to DNA.10
The motivation for focusing on studies of the effects of UV-A and UV-B, the solar UV wavelength
region, is provided by three observations:
1. Although many studies concerning the UV-C between 220 and 290 nm have been valuable in
elucidating some of the mechanism of cellular function, these wavelengths are environmentally
irrelevant.
2. Research has suggested that the nature of the primary and secondary chromophores, photoprod-
ucts and mechanisms for cellular response to solar UV appear, in some cases, to be very different
from those elucidated for wavelengths shorter than about 300 nm. For example, in contrast to
their marked sensitivity to UV-C, certain photosensitive human cell lines, such as Xeroderma
pigmentosum cells (XP), exhibit the same sensitivity to UV-A as normal cells.23–27
3. Solar UV impacts a large number of important bioresponses, such as skin cancer, plant growth,
cellular survival and mutagenesis, etc. It is also the region where many UV laser sources are
currently available. It is hoped that research in this area will ultimately reveal the nature of
biological responses to a portion of the radiation present in a normal environmental setting.
In addition, certain factors that may be involved in cell killing by UV-A, such as the disruption of the
cellular cytoplasmic microtubule complex observed by Zamansky and Chou,28 are not evident at shorter λ.
FIGURE 4.9 The UV-A, UV-B, UV-C action spectra for human cell killing, mutagenicity, DNA strand breaks, and
DNA-protein cross-links. These are mostly from Ref.30, with the exception of the AS for mutagenicity, which is
averaged from several reports. Note that all of these spectra diverge from the absorption spectrum of DNA in the
UV-A.
Also, glutathione seems to protect cells from the deleterious effects of UV-A, suggesting that at least some
UV-A damage is mediated via radical formation.29 The apparent lack of a close correlation between action
spectra for biological end points and UV-A-induced DNA damages (SSB, DSB, and DNA-to protein
cross-links) in human cells (Figure 4.9) can be explained in part by repair (see below) of these lesions.30–34
Two studies looked into the repair of SB induced by sunlamp irradiation. Roza et al.27 reported the
complete repair of all SSB within 60 mm of repair time (similar to the repair kinetics of x-ray-induced
SSB) in primary human fibroblasts and concluded that the lesions are irrelevant for cell lethality. Holm-
berg et al.35 found biphasic SSB repair in primary human lymphocytes exposed to UV-A — a fast (1
hour) and a slow (several hours) component, not observed by Roza et al.27 What is best known about
the effect of solar UV on living systems is that the nature of the responses is complex. At present, it is
not possible to document accurately the events that occur between photon absorption and biological
response. It appears that a large and varied number of chromophores are involved, some of which may
act only as intermediates. The nature of the final photoproducts is also unclear, as is their role in such
important events as cell death and mutation. How solar photodamage is processed or repaired is almost
unknown. That both UV-B and UV-A can cause cell death and mutation is well established, but the
mechanisms involved, especially in the UV-A region, are still obscure. Initial results that possibly link
certain endogenous chromophores with some cellular responses are promising. Little is known about
the synergistic or antagonistic effects of polychromatic exposure, especially mixtures of UV-B and UV-
A, which have such different properties, and essentially nothing is sure about such interactions for the
full spectrum of natural solar radiation. There is strong evidence that the mechanism of killing is not
the same for these different λ regions and that the initial damage may reside in a chromophore other
than DNA. That is, the lack of agreement between those bioeffects in the UV-A and DNA absorption
also seems to show that the primary chromophore(s) for mutagenesis is a non-DNA sensitizer, with
energy levels that match the incident photon energy, which acts as an intermediate in relaying the energy
to DNA. The identity of such chromophores has eluded all efforts to find them thus far.
4.7 Effects of UV on Cells

A considerable body of work that measures some of the bioresponses of cells to UV has been accomplished.
Several major areas of effect will be noted.
4.7.1 Cell Death

The most frequently measured cellular response to UV is cell death, usually assayed by measuring the
ability of the cell to form a multicellular colony. A large number of studies have shown that cell killing
is mainly a response to DNA damage. For example, pyrimidine molecules can absorb UV photons,
become excited, and interact to form dimers (Figure 4.6). These dimers are not recognized by the
reproductive apparatus of the cell and can prevent cell proliferation. Other DNA photoproducts are also
formed, including pyrimidine (6-4) pyrimidone, especially in the UV-A. It is also possible to produce
breaks in the DNA molecule backbone, either by direct or indirect effects sometimes involving the repair
of the photoproduct (see below). DNA can also link co-valently to protein molecules. These cross-links
are very stable and detrimental to the cell.
Cell survival after exposure to UV depends on a wide variety of conditions. Rapidly dividing cells are
usually more sensitive than quiescent cells. Pigments shield the DNA of some cells to UV exposure. Cell
geometry is important and can change during the cell cycle and under different irradiation conditions
(e.g., cells adhered to plastic petri dishes or grown in solution).11 A possible factor in cell killing by solar
UV is the disruption of the cellular cytoplasmic microtubule complex (cytoskeleton), which is important
for normal cell growth.28 Micro-tubule dissembly was observed after treatment with UV-A (but not UV-
B); this process could be the causative factor or one of many contributing factors in cell death. Microen-
vironmental conditions, including effects on non-UV-transparent buffers used in experiments and the
complex array of intercellular moieties in living tissue, must also be considered.
A comparison of the actual exposures required to kill various cell lines shows marked variations. For
example, normal mammalian cell lines exposed to broadband UV-A can differ in sensitivity by an order
of magnitude.26–30 The most probable cause of these differences, if rigorous spectral purity and comparable
dosimetry are adhered to, could be that each cell line may harbor different kinds or amounts of endog-
enous photosensitizers. Cells lacking efficient repair systems can be more sensitive than normal cells.
Depletion of endogenous glutathione markedly sensitizes human skin fibroblasts to killing by both
UV-B and UV-A, but not UV-C.29 The degree of glutathione protection approaches that derived from
excision repair (below). Glutathione protection confirms the indirect nature of UV-A lethality, suggesting
that a major fraction of UV-A damage, and also (surprisingly) UV-B damage, may be caused by radical
effects in photosensitized reactions of photodynamic action.
4.7.2 Cell Mutation

Anytime the genetic apparatus, especially the DNA molecule itself, is damaged, there is the possibility of
a mutation’s occurring. All UV wavelengths are mutagenic if a high enough exposure at that wavelength
reaches the DNA or if other chromophores absorb the photon and transmit the damage to the DNA
molecule. As is the case in cell killing, substantially higher exposures are required in the UV-B, and even
higher in the UV-A, to affect cells to a degree comparable to the effects derived from exposure to UV-C
(Figure 4.9). As is the case with cell death, different human cell lines may require different exposures to
any given UV wavelength to sustain similar levels of mutation This is especially true for human cells
derived from patients with certain photosensitive diseases such as XP, which results from a deficiency in
excision repair (below). These different responses do not necessarily extend over the whole UV range.
For example, Keyse et al.23 showed that a line of XP cells hypersensitive to UV-C was no more sensitive
to UV-A than were normal human cells. It is also speculative to extrapolate to human cells the mutation
rates determined with other cell types, e.g., in general, rodent cells are more sensitive to mutation by
UV, possibly because many rodents are nocturnal and are exposed to little, if any, UV. Human cells are
more proficient than frog cells in repairing solar UV lesions. Marsupial cells seem to have a sensitivity
between that of rodent and human cells. There is also one report that the wavelength 334 nm was non-
mutagenic in human epithelial cells.30 These authors carefully raised the exposure as high as 80 KJm–2,
an exposure that was mutagenic at 365 nm, and still could detect no level of mutation. Other cell lines
are mutagenic at 334 nm. These and other results with rodent and human cell lines show that it is not
possible to accurately predict the degree of mutation for any wavelength in the UV region. Lack of
correspondence with the spectrum of DNA in the UV-A (Figure 4.9) again suggests that the primary
chromophore(s) for mutagenesis in that region is a nonDNA sensitizer. Spectral studies show that the
yield of mutagenesis, for any given fluence, varies with wavelength but not in a manner consistent with
the involvement of any apparent specific primary chromophore. Further, different cell lines may harbor
different primary chromophores. Once the critical damaging chromophores are identified, it might be
easier to determine which photoproducts eventually lead to mutation at long UV wavelengths.
Broadband polychromatic sources in the UV-B and UV-A regions are also capable of causing mutation,
although it is difficult to know if such mutation induction is due to a small moiety of highly efficient
short wavelength or a large moiety of longer wavelength radiation. In the study of Hitchins et al.,36
mutation was observed even though no radiation of λ less than 340 nm was present.
4.7.3 Membrane Effects

Membranes, especially if they contain molecules that act as dyes and absorb UV, are also sensitive to UV.
Ion channels are affected, as is the respiratory system. In some cases, leakage of internal biomolecules
has been reported. Of course, it is imperative that the integrity of the membrane be preserved. How
much any of the above damages contribute to cell death is not presently known, but certain transport
problems could, if not kill, at least hinder a cell from functioning properly.17
UV-A radiation, at equitoxic exposures, seems to be more effective in damaging membranes. For
example, murine lymphoma cells lysed, and more than half of the remaining cells showed membrane
damage if exposed to UV-A levels that killed 90% of the cells.37 Similar results were obtained with sheep
red blood cells. Visible and infrared radiation had no lytic effects. However, human lymphoma cells
showed membrane damage but no lysis at similar exposures. Whether any of this relates to UV-A
cytoskeleton damage28 is not yet clear. But affecting membrane function and continuity by UV exposure
can serve both research and therapeutic purposes. As shown in Figure 4.5, short UV wavelengths (e.g.,
the ArF laser output at 193 nm) have such limited penetration into a single cell that only the membrane
and areas close to it are exposed. Cells damaged by membrane effects in this region should not be
susceptible to mutation, as the genetic material is not exposed. Cells damaged near the peak of DNA
absorption (260 nm) would be vulnerable to genetic change. At longer wavelengths, the situation is more
cloudy. All UV wavelengths are mutagenic (even 193 nm if naked DNA is exposed). The UV-B and UV-
A regions are useful experimental tools because they penetrate deeper into tissue. How a mutagenic risk
is estimated, however, depends on a large number of parameters. Low power level laser radiation, i.e.,
levels below which thermal damage predominates, may be useful therapeutically but could be mutagenic
to the surviving cells or disrupt cellular membranes.
4.8 Complex Responses of Tissues
4.8.1 Immune Effects

Perhaps the most comprehensive effect of UV on animals (including humans) are on the immune
response. Exposure to UV can compromise certain functions of the immune system, lowering an animal’s
ability to combat invasion by foreign substances and leading to an increase in infectivity, the spreading
of disease, and the efficacy of vaccination against disease. Currently, effects on the immune system have
been shown to be mainly due to exposure to UV-B. Whether UV-A exposure alone can elicit immune
suppression is still somewhat in doubt. UV damage to the immune system may explain why certain viral
infections, such as Herpes simplex, can recur in an individual who already has circulating antibodies
against the virus. Solar UV exposure can cause herpes recurrences in individuals previously infected with
the virus. The infectivity of the virus in individuals who do not harbor it in a latent form may also be
increased by UV exposure. Somewhat similar results have been obtained with HIV viruses.
The target chromophores for these immune effects are still questionable. However, one likely candidate
is urocanic acid,38 which, after absorption of UV, may initiate a chain of molecular events that ultimately
damage DNA. Figure 4.10 shows an action spectrum in mice for the immune response known as delayed
contact hypersensitivity.39 This spectrum has been shown to follow the absorption spectrum of urocanic
acid, which has a much higher extinction coefficient in the UV-B than does DNA. More recent results
further implicate this compound as at least one of the chromophores in the immune system’s response
to UV. Those are detailed in this book.
A recent study of human T-lymphocytes found them to be 20-fold more sensitive to UV-B radiation
than are human fibroblast cells.40 More alarming, naked T-lymphocytes cells in culture could be killed
by just a 1-minute exposure to sunlight. Even though little UV-B penetrates to capillaries in the skin,
extracapillary lymphocytes could receive lethal exposures. How this relates to the role of UV in immune
system suppression is still unknown. Sunlight can affect immune responses before it causes sunburn.
It has also been shown that UV radiation can accelerate the death of mice challenged by a lethal
infection of C. albicans.41 Here, the timing of the UV exposure was crucial, but the mechanism for the
effect was unknown. It seems clear that these and other effects on the immune system are widespread
and that the field of photoimmunology42 is evolving rapidly. These effects may turn out to be the most
damaging component of the human response to UV. Conversely, it may be possible in the future to
control some adverse immune reactions in humans by careful exposure to certain λ of UV.
Table 4.8 lists the predominant types of damage to cells by exposure to UV from different regions of
the spectrum. It is important to remember that these damages reflect the degree of involvement of each
process in cellular response. For example, a UV-C exposure that will kill a cell may also cause some
membrane damage, but the latter is a minor effect because the cell is killed by a different mechanism.
Note that VUV and UV-A both damage membranes, the former due to the limited penetration of VUV
into the cell (Figure 4.5), the latter to absorption of UV-A by components of the cell membrane. It
FIGURE 4.10 An action spectrum for photocarcinogenesis in mice, 43 and an AS for delayed contact hypersensitivity
(an immune response) in mice.39
TABLE 4.8 Predominant Types of Damage to Cells and Tissues by UV Region

UV Region Major Damage
Vacuum UV, 10–190 nm Surface effects; membranes; ionizations.

UV-C, 190–290 nm DNA altered; cell death; mutation; proteins; respiration; erthyma; carcinogenesis; immune
effects.
UV-B, 290–320 nm Transition region between UV-C and UV-A effects; solar UV-B causes cataract, erythema,
carcinogenesis, immune supression.
UV-A, 320–380 nm Various chromophores; indirect damage to DNA; repair systems; membranes; photoaging.
Note: All UV regions can cause cell death; the UV-A-B-C can also induce mutation, inhibition of respiration, and immune
responses, the exposures required are highly dependent on λ.
should also be reaffirmed that UV-A, UV-B, and UV-C are all mutagenic if enough exposure is given.
Because of the absorption properties of pigments, lasers can be aimed at specific pigmented targets,
e.g., melanin in melanocytes, hemoglobin in red blood cells, and exogenous drugs in photodynamic
treatment. For example, by laser exposure, it is possible to disrupt melanosomes within a melanocyte
without disrupting the cell.
4.8.2 Carcinogenesis
All known mutagens are potential carcinogens. The role of solar UV in human skin cancer has long been
established.4 Squamous and basal cell carcinomas are believed to be the result of chronic UV exposure.
The expression of these cancers is highly dependent on skin type and individual predisposition.
Figure 4.10 also shows the AS for carcinogenesis and contact sensitivity in mice. The carcinogenic data
is mainly from the analysis of De Gruijl and van der Leun43 and incorporates 12 separate studies. This
AS shows major effects in the UV-C and UV-B, and falls off rapidly in the UV-A, with a minor peak at
about 380 nm. However, due to the large preponderance of UV-A compared with UV-B in solar radiation
(about a factor of 35), the contribution of UV-A to carcinogenesis is significant. As shown, however, the
major cause of UV-induced cancer is the UV-B component of sunlight.
The extent of the role of UV in melanoma is still debatable. These skin cancers are often lethal if not
detected early after onset, and appear to be, at least partially, the result of acute exposures to sunlight,
especially in younger people. These sun-burning episodes may lead to the onset of melanoma several
decades after the exposure. It needs to be pointed out that melanomas often appear on areas of the skin
that are not usually exposed to UV. How, or even whether, these melanomas are associated with UV is
unknown, but one possible mechanism may be damage to the immune system. The long delay in the
response is not explained. Work on a UV AS for the production of melanoma in fish shows that UV-A λ
contributes a considerable amount of damage.
4.9 Repair
Complicating any analysis of UV effects on cells and tissues is the fact that cells have evolved efficient
molecular mechanisms to repair at least a portion of the damage caused by UV radiation.44 Usually, but
not in all cases (e.g., direct splitting of dimers by short UV photons), cells damaged by UV-C can be
repaired by UV-A (or even visible) radiation if an enzyme called the photoreactivating enzyme is present.
In the presence of UV-A, this enzyme splits the dimers caused by UV-C and UV-B radiation back into
monomers (Figure 4.6). A current controversy is whether this occurs to any real extent in human skin.45,46
High UV-A exposures can damage the repair system of the cell.
In addition to these photon-mediated repairs, a process known as excision repair can occur after UV
exposure in the absence of any light. Again, the integrity of the DNA molecule may be restored by a
complicated removal of the damage and resynthesis of an exact copy of the predamaged molecule. Other
repair processes exist and the extent and efficiency of each is still somewhat undetermined in human
tissue. For a good recent review see Sage.47
4.10 General Effects

The situation for UV exposure of cells and tissues is complex. A variety of active, quiescent, and dead
cells present themselves for irradiation. These cells may have a varying amount of pigmentation and
hence be differentially shielded from UV. Certain cells, somewhat dependent on their activity, may be
able to repair some of the damage caused by UV, in some cases using photons of other UV wavelengths.
Cells from certain diseased patients (e.g., XP) lack sufficient quantities of these enzymes for effective
repair. If the radiation reaches the blood, then blood-borne components, including cells, could absorb
enough UV and also be damaged. The final result of all this interplay may not be clinically observable
over the short term — or ever.
It is not unreasonable to expect a variety of uses in the future for targeting certain cells or tissues to
monochromatic laser exposure. For example, an oral or a local dose of a proper photosensitizer that
absorbs long wavelength UV can be given. The laser is then positioned (either within or outside the
body) to irradiate a well defined area of tissue (or even circulating cells), with the laser λ chosen matched
to the peak absorption of the photosensitizer. In this manner, only the targeted tissue is exposed. Such
photosensitizers can kill cells. If a drug being used for other purposes is also a photosensitizer, then a
warning advising patients to limit their solar exposure is usually listed in the descriptive folder packaged
with the drug sample.
It is important to estimate the degree of penetration of the UV from the source into the targeted tissue.
Table 4.7 shows a rough estimate of the extent of penetration of UV through human epidermis. Any such
measurement is highly dependent on skin type and pigmentation and will even vary among individuals
of the same skin type. The table shows data averaged from two sources.12,48
Table 4.9 is an abbreviated list of some of the uses and effects of UV in biomedicine. Erythema is
a common skin response mainly due to UV-B exposure, whereas photoaging is mainly a UV-A
phenomenon. The treatment of various skin disorders, such as psoriasis, with drugs and UV is now
common worldwide. But the surgical applications of UV rely mainly on the availability of lasers. These
applications range from the sculpting of deformed corneas by microablation with 193 nm laser
radiation to the ablation of arterial plaque by 308 nm laser radiation. The benefits and risks of each
of these procedures are being studied, but these and other treatments hold promise for an even wider
use of lasers in medical treatment.
TABLE 4.9 Some Examples of the Uses and Effects of UV in Biomedicine

Treatment for Surgical use Other
Acne vulgaris Glaucoma Erythema

Psoriasis Angioplasty Vit. D synthesis
Mycosis fungoides Corned sculpting Photoaging
Eczema Endoscopy Photoimmunology
Vitiligo Keratogomy Photodynamic therapy
4.11 A Perfect Laser for UV Photobiological Studies

The wishes and requirements for the perfect UV radiation source may well be different for the majority
of the photobiological community, when compared with the needs of physicists and physicians. As listed
in Table 4.2, in many cases a photobiologist would prefer a high total energy source and would not be
satisfied with a source with high peak power but low total output power. Of course, those scientists
studying molecular effects such as time-resolved spectroscopy, and those physicians interested in ablating
tissue, would require high peak power. In fact, some uses of lasers in therapy and diagnosis will require
pulsed sources in which the exposure can be more easily controlled in stepwise increments. It is important
to observe whether such high power lasers cause unwanted multiphoton effects, which may be of use in
the study of certain molecular phenomena, but the natural exposure of cells and tissues usually occurs
in a continuous manner and depends on the availability of certain chromophores for absorption. Except
for isolated studies, it would also be preferable to have a tunable source for action spectra generation,
and for exposure of different cells and tissues, especially in combination with different photosensitizers.
Although it is wishful thinking, the breadth of tunability should be as wide as possible over the UV
region, say from 200–380 nm. Laser spectral purity is often more stringent than needed for the irradiation
of biomolecules and cells, and already exceeds the requirements for many photobiological uses, as
biomolecules have broad absorption spectra and cannot “resolve” photons to more than a few nm. The
laser should also be simple to operate. Ideally, such a “photon box” should easily plug into an outlet,
have an on–off switch, a λ selection dial, and an intensity dial. It should require few skills to operate and
never break down. Athough the availability of such a source is still a fantasy, probably only with lasers
can it even be approached.
4.12 Useful Review Texts

Several good texts to introduce scientists to the various areas of photobiology are available. Two
books by Jagger5,17 provide both an introduction to UV photobiology and an analysis of solar UV
effects. Smith’s Photobiology49 is a general introduction that includes a fuller spectral range. Douglas
et al.50 is also a good general review. Three texts confine themselves mainly to UV-A effects,12,51,52
all incorporating many biochemical applications. The rapidly emerging field of photoimmunology
is summarized by Parrish et al.42 Molecular photodamage and repair of DNA is detailed in Hanawalt
and Setlow44 and Simic et al.31 Because of renewed interest, partially due to the depletion of
stratospheric ozone and the consequent expected rise in UV-B reaching the biosphere, and the
increased use of UV in diagnostic and therapeutic procedures, many shorter monographs that
address more limited areas of photobiology are appearing.53 Recent books and review articles on
photobiology are cited in.54,55
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42. Parrish, J.A. M.L. Kripke, and W.L. Morison, in Photoimmunology, Plenum Press, N.Y. (1983).
43. de Gruijl, F.R. and J. van der Leun, Action spectra for photocarcinogenesis, in Biological Responses
to UV-A Radiation, Ed. F. Urbach, Valdenmar Publishing, Overland Park Kansas (1992).
44. Hanawalt, P.C. and R.B. Setlow, Molecular Mechanisms for Repair of DNA, Parts A and B, Plenum,
New York (1974).
45. Ley, R., Photoreactivation in tissues, in Biological Responses to UV-A Radiation, Ed. F. Urbach,
Valdenmar Publishing, Overland Park, KS (1992).
46. Sutherland, B.M. et al., Pyrimidine dimer formation by UV-A radiation: implications for photo-
reactivation, in Biological Responses to UV-A Radiation, Ed. F. Urbach, Valdenmar Publishing,
Overland Park, KS (1992).
47. Sage, E., Distribution and repair of photolesions in DNA: genetic consequences and the role of
sequence content, Photochem. Photobiol. 57, 163-174 (1993).
48. Everett, M.A. et al., Penetration of epidermis by ultraviolet rays, Photochem. Photobiol. 5, 533-542
(1966).
49. Smith, K.C., The Science of Photobiology, Plenum, New York (1977).
50. Douglas R.H., J. Moan, and G. Ronto, Light in Biology and Medicine, Plenum, New York (1991).
51. Urbach, F. and R.W. Gange, The Biological Effects of UV-A Radiation, Praeger, New York (1986).
52. Urbach, F., Biological Responses to UV-A Radiation, Valdenmar Publishing, Overland Park, KS
(1992).
53. Wilson, B.C., Ed., Lasers in medicine, Photochem. Photobiol. 53, special issue (1991).
54. Urbach, F., 25th Anniversary Symposium, Landmarks in Photobiology, Photochem. Photobiology.
655, 1055-1515.
55. Coohill, T.P., Photobiology for the 21st Century, Valdenmar Publishing, Overland Park, KS (2001).
5
The Physics
of Ultraviolet
Laser Ablation
5.1 Introduction ....................................................................... 109

Laser Characteristics • Material Characteristics
5.2 Deposition of Ultraviolet Radiation in
Organic Materials.....................................................................113
More Advanced Considerations
George H. Pettit 5.3 Target Decomposition........................................................ 120
Summit Autonomous 5.4 The Ablation Plume ........................................................... 122
5.5 Repetitive Irradiation ......................................................... 128
References ...................................................................................... 129
5.1 Introduction
In 1982, two articles describing the ablation of organic material by excimer lasers appeared in the scientific
literature.1,2 Those initial studies demonstrated that intense pulses of ultraviolet radiation precisely etched
submicron layers from synthetic polymer surfaces and caused negligible thermal damage to the remaining
target. Such microscopic accuracy generated much interest in the materials processing community, as
well as in several medical disciplines. Within 1 year, argon fluoride (ArF, λ= 193 nm) excimer laser
ablation was being explored as a means of reshaping the surface of the cornea to correct for vision defects.3
Soon thereafter, xenon chloride excimer laser radiation (XeCl, λ = 308 nm), which could be transmitted
by a flexible fiberoptic catheter, was studied as a tool for clearing atherosclerotic blood vessel obstructions
in a noninvasive manner.4 Over the ensuing years, clinical interest in pulsed UV laser ablation of tissue
has continued to grow, as discussed elsewhere in this book.
In this chapter, the physical mechanisms underlying the ablation process are described. Tissue ablation
is the discrete vaporization of a microscopic quantity of biological material by a pulse of laser radiation.
It is a complex event encompassing multiple phenomena occurring on a short (nanosecond to micro-
second) timescale. To provide an organizational framework, the material is partitioned into six categories,
which are presented in roughly chronological order over the course of an ablation occurrence. Within
several of these six areas, the material is further subdivided into two sections. The first part deals with
the subject in a general qualitative sense, while the second explores it on a more quantitative level. While
this outline facilitates the discussion, it is an essentially arbitrary structure. Several of the delineated
events, such as the absorption of laser radiation and the decomposition of the target material, occur
simultaneously, and undoubtedly impact on each other. These interrelationships are important aspects
of the ablation process and are discussed in some detail.
0-8493-1146-2/02/$0.00+$1.50
Although the theme of the text is the medical application of laser radiation, UV laser ablation studies
involving nonbiologic as well as biologic organic material are discussed here. A wealth of data exists
in the scientific literature on the topic of synthetic polymer ablation, due to numerous important
applications in the microelectronics industry and elsewhere (e.g., calibration of medical laser systems).
In addition, the chemical and physical properties that facilitate analysis of the laser–target interaction
are better established for man-made plastics than for tissues. Because many aspects of the process are
similar for both polymers and tissue, much useful information can be gleaned from these investigations.
Where significant differences are known to exist between biologic and nonbiologic substrate ablation,
these are expressly stated in the text.
5.1.1 Laser Characteristics

Although the specific lasers mentioned in the introduction are both excimer systems, the ablation
mechanisms described in this chapter apply generally to any device fulfilling three criteria:
1. The laser must produce radiation at an ultraviolet wavelength. The relevant part of the electro-
magnetic spectrum extends from about 180 nm to 380 nm.
2. The laser output should be in the form of submicrosecond pulses of radiation.
3. When the laser is delivered to an ablation site, the energy per pulse must be sufficient to produce
a fluence (energy per unit area) above some threshold. The exact value of this ablation threshold
fluence is both material- and laser wavelength-dependent.
The importance of each of these characteristics is now considered in turn.
Because the energy associated with an individual photon increases with decreasing wavelength,
photon energies at short ultraviolet wavelengths are substantial. In early analysis of the photoablation
process, it was hypothesized that these energetic UV photons caused direct “bond-breaking” of the
molecular chains in the organic target, and that the photochemical fragmentation vaporized material
with negligible heating (vibrational excitation).5 This mechanism may, in fact, play a significant role
in laser ablation at wavelengths below ~200 nm, where photon energies are significantly greater than
the typical molecular bond strengths in organic polymers. However, similar ablation effects are
observed for longer ultraviolet laser wavelengths where the photon energy is insufficient to break
organic bonds.4,6,7 There now appears to be general agreement among researchers that the photoabla-
tion phenomenon at most ultraviolet wavelengths is primarily due to multiple-photon processes,
including localized heating.6–9 The absolute importance of single-photon bond-breaking to the event
is still debated.
A separate feature of ultraviolet radiation universally considered to be important for laser ablation is
that it is very strongly absorbed in organic targets (i.e., tissue or synthetic polymers). Reported values
for the 1/e penetration depth of 193 nm ArF excimer laser radiation in corneal tissue range from 0.3 to
4 µm,10,11 and only slightly higher in the plastic polymethylmethacrylate (PMMA).12 This means that,
when a laser pulse strikes an organic target, the radiation is deposited in a thin surface layer of the
substrate, resulting in a localized region of very high deposited energy density. Whatever effect the laser
pulse has on the material, i.e., either bond-breaking or vibrational heating, occurs in that narrow
superficial volume.
Laser alteration of the material is also confined by the submicrosecond pulse duration. Simply stated,
laser energy is absorbed by the target volume much faster than it can diffuse as heat into the adjacent
bulk. The competition between laser absorption and heat diffusion is shown schematically in Figure 5.1.
One measure of the rate of heat diffusion out of a target region is the thermal relaxation time τt, defined
as the time required for the excess temperature in the target to fall by half. If the laser penetration depth
is on the micron scale, then for virtually any biomedical application, the lateral dimensions of the
absorbing volume will be several orders of magnitude larger than the depth. Heat diffusion out of the
heated region is virtually unidirectional, into the deeper target. Under these conditions, the thermal
relaxation time can be estimated by:13
The Physics of Ultraviolet Laser Ablation 111
incident
laser pulse
target
surface
absorbing
region
heat
diffusion
FIGURE 5.1 The fundamental laser–tissue interaction, which is central to the ablation process. An incident laser
pulse is absorbed in a surface layer of the target tissue. After absorption, the energy originally contained in the pulse
can diffuse as heat into the surrounding tissue. By delivering the energy as fast as possible, i.e., in a very short pulse,
and choosing a laser wavelength where the tissue is highly absorbing, the laser energy can be initially confined to a
small region of the tissue and cause vaporization.
1
τ t = ------------
2
, (5.1)
4α β
where α is the absorption coefficient of the target at the laser wavelength and β is the thermal diffusivity
of the material. For aqueous biological tissues, values for α in the ultraviolet typically lie between 102–104
cm-1 (UV absorption in synthetic polymers can exceed 105 cm-1), while β is on the order of 10-3 cm2 s–1.14
Therefore, the relaxation time for a target tissue volume will be ~100 µs or greater. A laser pulse more
than two orders of magnitude shorter than this relaxation time will be completely absorbed before any
significant thermal diffusion occurs.
If the fluence of the incident laser pulse exceeds some threshold, the third essential criterion, material
in the absorbing region, will decompose into smaller molecular weight products that are then ejected in
the ablation event. As indicated above, the precise value of the ablation threshold fluence is extremely
case specific. For example, the measured ablation threshold for ArF laser ablation of the cornea is slightly
less than 50 mJ/cm2 per pulse,11 while the threshold fluence for XeCl laser ablation of calcified arterial
wall tissue is approximately 3.0 J/cm2 per pulse.15,16 Practical considerations as to beam delivery and
useful target spot size dictate that clinical laser systems for these applications produce tens to hundreds
of millijoules per pulse.
The earliest pulsed UV lasers generally available for ablation work were excimer devices. An excimer
laser works by passing a controlled intense electrical discharge through a gas-filled cavity. The gas mixture
in the cavity has three components: a noble gas (argon, krypton, or xenon), a halogen (either fluorine
or chlorine), and an inert buffer gas (helium and/or neon). The electric discharge leads to the transient
formation of noble gas or halogen molecules, and the subsequent energetic dissociation of these unstable
molecules releases the ultraviolet photons that make up the laser output.
Excimer lasers typically produce pulses lasting 10–30 ns, although special versions of the XeCl laser
have been built in which the pulse is “stretched” to several hundred nanoseconds’ duration. This length-
ening of the pulse facilitates the efficient coupling of the 308 nm radiation into optical fibers,15 an essential
task for laser angioplasty. Such an increase in laser pulse length has been shown to not substantially affect
the tissue ablation achieved for constant-fluence pulses16,18 consistent with the orders of magnitude longer
thermal relaxation time.
Despite the excellent tissue ablation characteristics of excimer lasers, the fairly large size of these devices
and their requirement for pressurized halogen gases make them less than ideally suited for clinical use.
These limitations have prompted development of alternative pulsed ultraviolet sources. Currently, the
most advanced of these is the Q-switched nd:YAG laser. This solid-state laser produces intense (up to 1
joule or more) nanosecond pulses at a wavelength of 1.064 µm. By passing this intense IR radiation
through nonlinear crystals, it is possible to obtain ultraviolet pulses at 3 wavelengths: 355 nm, 266 nm,
and 213 nm. Laser radiation at 355 nm has been employed in experimental angioplasty work,19 while
213 nm radiation is being studied for corneal sculpting.20
5.1.2 Material Characteristics

What characteristics of the target material are important in the ablation process? Obviously, to be affected
by an incident laser pulse, a target must absorb some fraction of the pulse energy. Absorption in organic
material is due to specific molecular or submolecular features called chromophores. In biological systems,
the most common molecule is water, composing typically ~70% of the total tissue mass. While absorption
by water is stronger in the ultraviolet than in the visible, over the range of wavelengths considered here,
the absorption coefficient for water at physiological temperatures does not exceed 10 cm-1.21 Therefore,
water is not usually considered a significant chromophore for UV laser pulses, although it has recently
been hypothesized to play an important absorptive role at elevated target temperatures.22
The next most prevalent component in tissue is the organic fraction, composing ~25% of the total
wet weight. This grouping includes the proteins, lipids, nucleic acids, etc., that make up the various
cellular and extracellular structures. Such biochemical entities are typically rich in ultraviolet chro-
mophores. Consequently, most of the energy in an incident UV laser pulse will be absorbed by the organic
tissue component.
Protein, the dominant organic entity in tissue, consists of a chain of amino acids connected by peptide
bonds, as shown in Figure 5.2. Two ultraviolet chromophore types exist in the macromolecule — the
amino acid side chains and the peptide bond itself. The identity and concentration of the different side
chain absorbers vary widely between proteins, while the peptide bond is always present in abundance.
The absorptive strength of these different entities is conventionally reported in the biochemical literature
as the molar extinction coefficient or molar absorptivity, e, reported in units of cm–1 M–1.
Of the 20 common amino acids, three have side chains incorporating a 6-carbon aromatic ring:
tryptophan, tyrosine, and phenylalanine. Benzene itself is a well-known ultraviolet chromophore, with
a strong absorption peak at 198 nm (ε = 8000 M–1 cm–1), and a second weaker absorption band centered
around 255 nm (e = 230 M–1 cm-1).23 When the aromatic ring structure is incorporated into an amino
acid, these characteristics are essentially retained. The molar extinction coefficients for the aromatic
amino acids at the 193 nm ArF laser wavelength lie between 2–5 × 104 M–1 cm–1.24 Tryptophan, the
largest, most highly conjugated amino acid, also exhibits a significant absorption band over a broad
wavelength region centered around 275 nm.25 Tyrosine absorbs at these wavelengths as well, although
at only one fourth the magnitude of tryptophan, due to a less complex side chain.25 Phenylalanine,
the simplest aromatic amino acid, exhibits only very weak absorption in the vicinity of 255 nm.25
Therefore, a high concentration of aromatic amino acids confers a strong far ultraviolet absorption
capacity to a protein molecule, as well as moderate mid-UV absorption (dependent primarily on
tryptophan).
Proteins composed of nonaromatic building blocks still absorb significantly in the far ultraviolet, due
to the peptide bond. This molecular structure exhibits an absorption peak at approximately 185 nm,
which falls with increasing wavelength to essentially zero by ~240 nm.26 The molar extinction coefficient
for the bond at the 193 nm ArF excimer wavelength is 5.5 x 103 cm-1 M-1,24 roughly an order of magnitude
less than that for the aromatic amino acids. However, as this feature occurs once for every amino acid
along the protein chain, the number of peptide bonds in the macromolecule is always very large.
Therefore, far ultraviolet absorption by peptide bonds is important in all proteins, even those rich in
aromatic side chains.
Other organic biologic molecules, less prevalent than protein in most tissues, play a secondary role in
the absorption of a UV laser pulse. All exhibit some absorption in the far ultraviolet. The nucleic acids
(DNA and RNA) are also absorbing at mid-UV wavelengths between ~250 nm and 290 nm.24,26 This is
not the case with carbohydrates, which are essentially transparent to radiation above about 230 nm.26
Lipids, which primarily contain simple hydrocarbon bonds, are also essentially nonabsorbing throughout
most of the ultraviolet.
side peptide side

chain bond chain
X O Y O
N C C N C C
H H H H
amino acid amino acid

FIGURE 5.2 The essential molecular structure of protein, an important tissue absorber of ultraviolet laser radiation.
Proteins are biological polymers composed of multiple amino acid subunits linked by peptide bonds. The uniqueness
of each amino acid is entirely due to the side chain. In turn, the structure and function of the protein macromolecule
is dependent on the identity and ordering of the constituent amino acids.
The light-scattering potential of a target also affects its interaction with laser radiation. Tissue is an
inherently scattering medium at ultraviolet wavelengths due to the high density of refractive index
fluctuations in biologic structures. Because scattering in a material typically increases as wavelength
decreases, the phenomenon would be expected to be most pronounced at the shortest ultraviolet
wavelengths. However, since the absorption coefficient in tissue increases even more dramatically with
decreasing wavelength, scattering has the largest effect on laser pulse propagation in the near ultraviolet,
at wavelengths longer than ~280 nm. For example, the scattering coefficient for 308 nm radiation in
normal human aorta is 77 cm–1, while the absorption coefficient in the same tissue is only 33 cm–1.27
Thus, most of the attenuation of a forward directed XeCl laser pulse in aortic tissue is due to scattering
rather than absorption.
5.2 Deposition of Ultraviolet Radiation in Organic Materials

The first challenge in understanding the interaction of an ultraviolet laser pulse with an organic target
is to describe how the pulse energy is deposited in the tissue. A common starting point for such analysis
is Beer’s law, also referred to as Beer-Lambert law, which states that the incident laser pulse fluence F0
will be attenuated exponentially versus depth x in the material:
– αx
F ( x ) = F0 e (5.2)
where F(x) is the residual pulse fluence crossing x, and α is the measured absorption coefficient of the
target. (The absorption coefficient is also often noted by µa). A clear distinction should be made between
the absorption coefficient α, commonly used in the physical literature, and the molar extinction coeffi-
cient e introduced above. The molar extinction coefficient is defined as:23
F(x)
log  -----------
 F0 
ε = – ---------------------------
10
- (5.3)
Cx
where C is the molar concentration of the chromophore in question. Thus, for a single absorbing species,
α = e C log10(e).
Equation 5.2 is only partially applicable to the case of UV laser tissue ablation. The effects of light
scattering, which may be significant at long ultraviolet wavelengths, are not included in the equation.
In addition, α is assumed to be a constant equal to the measured low intensity value. Under intense
irradiation, the effective attenuation coefficient of the target may differ significantly from the small-
signal value and may change during the laser pulse.28,29 Such effects are due to multiple-photon
absorption and other nonlinear processes. Despite these shortcomings, Equation 5.2 can still be used
to derive a qualitative description of the laser pulse deposition that yields useful insight into the
ablation process.
To understand the effect of a laser on a target, one should know the amount of pulse energy deposited
per unit volume in the material. This quantity will also vary with x, and is defined here as E(x). As a
laser pulse penetrates into an absorbing medium, if scattering may be neglected, the fluence decrease per
unit depth is equal to the energy absorbed per unit volume:
dF – αx
E ( x ) = – ------ = αF 0 e . (5.4)
dx
Thus, like the pulse fluence, the deposited energy density also decays exponentially with depth. However,
note that E(x) is considerably more dependent on the target absorption coefficient α, than F(x) is.
Maximum energy deposition occurs at the surface (x = 0), where E(0) is equal to α F0.
On the basis of Equation 5.4, one can infer that the threshold fluence for ablation of a substance, Fth,
corresponds directly with a threshold deposited energy density, Eth = α Fth. Experimental evidence suggests
that Eth is an intrinsic material property, relatively independent of the ultraviolet laser wavelength.7,30 It
is logical to assume that, as the incident fluence F0 is increased above Fth material will be ablated to a
finite depth, d, where E(x) falls to Eth. A mathematical expression relating incident fluence to ablation
depth can be derived using these relations. By starting with:
E ( d ) = E th , (5.5)
and, substituting on both sides:
– αd
αF 0 e = αF th , (5.6)
we can arrive at the simple expression:
F
d = --- ln  ------0  .
1

(5.7)
α F th
Equation 5.7 predicts that the etch depth per pulse will increase logarithmically with fluence above
threshold. (Experimental laser etching results typically exhibit significant deviations from Equation 5.7,
for reasons discussed below.)
This theoretical framework can be used to explain several of the wavelength-dependent effects observed
in experimental ablation studies. For example, consider a tissue being irradiated by one of two pulsed
lasers operating at distinct wavelengths, λ1 and λ2, with absorption in the target being much higher at
the first wavelength (α(λ1) » α(λ2)). According to Equation 5.4, for an incident pulse of fluence F0, the
deposited energy density at the target surface will be much greater for the laser operating at the λ1
wavelength. Furthermore, the energy density will fall off much more rapidly with depth at λ1 than at λ2.
These effects are shown graphically in Figure 5.3.
In Figure 5.3a, the incident laser pulse is of relatively low fluence. However, since the deposited energy
density at the target surface is the product of fluence and absorption coefficient, the ablation threshold
is exceeded for the more strongly absorbed λ1 laser radiation, and material is removed to depth d1.
Therefore, the threshold fluence for ablation is lower at the more strongly absorbed laser wavelength.
This has been found to be true in virtually all ablation studies.31,32
In Figure 5.3b, the incident laser pulse is much more intense. The deposited energy density at the
material surface exceeds Eth for either laser wavelength, therefore, ablation occurs in both cases. Note
a: low fluence laser pulse
Deposited Energy Density

E1(x)
E2(x)
Eth
d1
Depth in Material
b: high fluence laser pulse

Deposited Energy Density
E1(x)
E2(x)
Eth
d 1 d2
Depth in Material
FIGURE 5.3 Deposition of two identical-fluence laser pulses at different wavelengths, λ1 and λ2, in a material which
absorbs much more strongly at λ1. Ablation will occur in the material to a depth where E(x) falls to some threshold
value, Eth. (a) Absorption of a low fluence laser pulse. Ablation only occurs for the λ1 laser wavelength. (b) Absorption
of a high fluence pulse. Laser radiation at either wavelength causes ablation, with the λ2 pulse removing material to
a greater depth.
that the etch depth achieved at λ2, the weakly absorbed laser wavelength, exceeds the depth produced
by the λ1 laser pulse (d2 > d1). As the incident fluence is increased still further, d2 will continue to
grow much more rapidly than d1. These trends are also borne out in experimental work; at laser
fluences above the threshold regime, a smaller target absorption coefficient results in a greater
dependence of etch rate on fluence, and, consequently, for most practical fluences, a significantly
deeper ablation depth per pulse.7,31,32
One other aspect of the laser–target interaction can be inferred from Figure 5.3b. Since the fluence in
the laser pulse is the same at both wavelengths, the integral of E(x) over all depths, i.e., the area under
either curve, is also the same. Note that, for the pulse at λ1, the vast majority of the laser energy is
absorbed in the ablation volume from the surface to d1. In the λ2 case, the average value of E(x) in the
ablation volume is much less, and a larger fraction of the pulse energy is deposited below d2, in the intact
target. The excess energy in the ablated region under high absorption conditions results in decomposition
of the original organic material into smaller molecular fragments. The ablation products for ArF excimer
laser irradiation of PMMA are, on average, significantly smaller in molecular weight than those for
krypton fluoride (KrF, λ = 248 nm) excimer laser irradiation, in part due to the 35-times higher absorption
coefficient at the 193 nm wavelength (α193 = 2.0 × 103 cm-1 versus α248 = 5.7 × 102 cm-1).31 The greater
residual energy left in the target under low absorption conditions is converted to heat,30,33 which may
contribute to damage of the site.
The concepts presented in this section are exemplified particularly well by comparing ArF and KrF
excimer laser ablation of corneal tissue. The cornea of the eye is composed primarily of collagen, which
accounts for ~70% of the dry weight of the tissue.34 Collagen is a somewhat unusual protein, in that it
is almost devoid of aromatic amino acids and absorbs ultraviolet radiation only through the peptide
10
Corneal Ablation Depth per Pulse (µm)

1
0.1
0.01
193 nm
248 nm
0.001
10 100 1000 104
2
Laser Fluence per Pulse (mJ/cm )
FIGURE 5.4 The reported ablation depth per pulse in cornea as a function of incident laser fluence at 193 nm and
248 nm, based on literature sources as cited in the text. The distinct ablation behavior observed at the two wavelengths
is largely attributable to the much larger tissue absorption coefficient at the shorter ultraviolet wavelength.
bond chromophores. Because of this, the absorption coefficient of the cornea is much higher at 193 nm
(between 2700 and 40,000 cm–1)10,32 than at 248 nm (210 cm).32 The ablation behavior of the tissue is
shown in Figure 5.4, based on published measurements of corneal etching by either ArF 32,33,35–41 or KrF32,35
excimer lasers. ArF laser ablation of the tissue results in a lower threshold and a slower increase in etch
depth versus tissue than does KrF laser ablation. In addition, KrF laser irradiation of the cornea is
associated with more damage to the residual tissue. Furthermore, an examination of the ablation plumes
produced at the two excimer wavelengths indicates much smaller ablation particles at 193 nm.42 All these
findings are consistent with the absorption analysis discussed above.
5.2.1 More Advanced Considerations
One limitation of the simple theory presented above is that it assumes that α is constant over the course
of the laser pulse. At ablative laser intensities, the optical properties of the target can change substantially
during nanosecond-timescale irradiation. For example, in the case of ArF laser ablation of cornea, a
detectable decrease in laser transmission has been observed for irradiation of microtome thin tissue
slices.29 Both transmission decreases and increases have been measured during UV-laser ablation of
various synthetic polymers.43–45 In this subsection, the theoretical description of the ablation process is
extended to analyze this dynamic (i.e., radiation-dependent) target absorption.
As an example, we will first consider the well-studied case of 248 nm KrF laser ablation of polyimide.
The measured relationship between polyimide etch rate and KrF laser pulse fluence is shown in Figure 5.5.
The discrete data points in the figure are taken from two experimental studies, one using a thin polyimide
film technique very accurate at low pulse fluences (∆9) and the other based on perforation of thicker
polyimide sheets by repetitive irradiation (o6). (The discrepancies between the two sets of data are most
likely due to unavoidable inaccuracies in the repetitive irradiation method at laser fluences just above
the ablation threshold.) The continuous line in the figure is the “blow-off ” theoretical relationship, based
on reported values for the ablation threshold fluence (28 mJ/cm2) and absorption coefficient (25 µm-1).7
Clearly, material is being removed to a significantly greater depth than predicted by the theory, particularly
at higher laser pulse fluences.
The transmission characteristics of polyimide have also been studied over this same range of KrF laser
pulse fluences, as shown in Figure 5.6.43 The vertical axis in the figure indicates the fraction of incident
KrF laser pulse energy transmitted through a 0.1 µm-thick polyimide film (1 = total pulse energy
transmission). The continuous horizontal line in the figure indicates the predicted transmission behavior
0.8
0.7
Ablation Depth per Pulse (µm)

0.6
0.5
0.4
0.3
0.2
0.1
0
10 100 1000 10000
Laser Fluence per Pulse (mJ/cm2)
FIGURE 5.5 The reported ablation depth per pulse in the synthetic polymer polyimide as a function of incident
laser fluence at 248 nm, indicated by discrete symbols, based on two literature sources as cited in the text. The
continuous curve indicates the ablation behavior predicted by simple “blow-off ” theory (Equation 5.7). The dashed
curve is based on a slightly more complex model that takes into account radiation-induced changes in the material
absorption coefficient (Equation 5.19).
0.8
Laser Energy Transmission
0.6
0.4
0.2
0
10 100 1000 10000
Laser Fluence per Pulse (mJ/cm2)
FIGURE 5.6 Measured transmission of a 0.1 µm-thick film of polyimide as a function of KrF laser pulse fluence
(see text). For ablative laser fluences, the transmission of the film increases substantially, due to radiation-induced
decreases in the target absorption coefficient. Curves in the figure are theoretical models of the absorption behavior,
as described in the text.
of the film, i.e., a constant ~ 8%, based on Beer’s law and the measured absorption coefficient of the
material. With increasing laser intensity, a progressively greater fraction of the incident 248 nm radiation
passes through the polyimide film.
To understand these two interrelated findings in Figures 5.5 and 5.6, we must analyze laser absorption
by the target in a slightly more detailed way.43,46 We do this using the simple two-level absorption scheme
shown in Figure 5.7. Consider an idealized organic target with a single population of chromophores, all
with identical absorption characteristics and present in the material at a density ρ. Initially, all chro-
mophores are in the ground state (0). Absorption of photons from an incident laser pulse will promote
some chromophores to the excited state (1). The densities of chromophores in the ground and excited
states at depth x in the material and at time t during the irradiation are noted as ρ0(x,t) and ρ1(x,t). The
1 ρ1(x,t)
l(x,t)
σ1
0 ρ0(x,t)
FIGURE 5.7 Theoretical scheme for the single-photon absorption of ultraviolet laser radiation by an organic
substrate. Material chromophores, initially in the ground state (0), are promoted to the excited state (1) by absorbing
single photons from the incident laser pulse. The excited chromophore state may have substantially different absorp-
tion properties from the ground state.
affinity of a single ground-state chromophore for a photon at the laser wavelength is expressed as the
absorption cross-section, σ1, which has the units of area. (The cross-section in cm2 for an individual
chromophore can be directly calculated from the molar extinction coefficient, when reported in M-1 cm1,
simply by multiplying by 1.66 × 10-21.) The product of the initial ground-state chromophore density
(ρ0(x,0) = ρ) and the absorption cross-section must be equal to the material absorption coefficient:
ρσ 1 = α (5.8)
Under conventional, i.e., low-intensity irradiation, the density of photons absorbed per volume of mate-
rial is much less than ρ and absorption in the material is accurately described by a constant α. However,
at high laser intensity, this is not necessarily the case. For polyimide, the density of building-block imide
monomers in the intact material is about 2.3 x 1021 cm-3. Each monomer is rich in double bonds and other
features known to be strongly absorbing in the ultraviolet,6 so that the number of 248 nm chromophores
per monomer may be as high as 20. Therefore, ρ in this situation can be estimated to be 4–5 × 1022 cm–3.
Using the above stated values for α and Fth in Equation 5.4, we can estimate Eth to be about 7,000 J/cm3,
or 9 × 1021 absorbed 248 nm laser photons per cm3 (based on the energy associated with each photon being
8 × 10-19 J). Therefore, just at the ablation threshold, the density of laser photons deposited in the surface
region is almost one fourth as large as the chromophore density. This fraction becomes progressively more
substantial as the incident pulse fluence is increased above threshold.
We therefore must consider excited state chromophores in analyzing the radiation transport process.
Many plausible suppositions can be made about the first excited state in Figure 5.7. For example, the
chromophore might quickly (relative to the pulse duration) return to the ground state through either
radiative or nonradiative decay. That mechanism would allow for some laser energy deposition in the
material without appreciably altering the absorption, not the effect we are presently trying to describe.
In this particular instance, we will assume that each chromophore promoted to the excited state is
permanently bleached by absorption, as would occur with rapid dissociation of the excited state entity
into new nonabsorbing species.
If the laser flux (photon per area per time) crossing depth x in the material is given as I(x, t), then
the density of ground and excited chromophore states in the material will evolve over time as:
∂ρ 0 ( x, t ) ∂ρ 1 ( x, t )
- = – σ 1 ρ 0 ( x, t )I ( x, t ) .
--------------------- = – -------------------- (5.9)
∂t ∂t
I(0,t) of course, is the incident photon flux, determined by the temporal characteristics of the laser. In
a similar fashion, the spatial attenuation of the incident photon flux will be:
∂I ( x, t )
----------------- = – σ 1 ρ 0 ( x, t )I ( x, t ) (5.10)
∂x
Equations 5.9 and 5.10 can easily be solved. Integration of 5.9 yields:
t
  
  ∫
ρ 1 ( x, t ) = ρ  1 – exp  – σ 1 I ( x, t′ ) dt′ 

(5.11)
0
Substituting Equation 5.11 into 5.10 gives:
t
∂I  
∂x  ∫
----- = – ρσ 1 I ( x, t ) exp  – σ 1 I ( x, t′ ) dt′ .

(5.12)
0
Because the duration of excimer laser pulses is typically several orders of magnitude less than the
interval between pulses, Equation 5.12 can be integrated from time zero to infinity to obtain the atten-
uation for the total pulse photon density S(x), where:
S(x) = ∫ I ( x, t ) dt′ . (5.13)

0
S(x) times the photon energy is the fluence crossing depth x in the material. Integration of Equation
5.12 then yields:
∞ t
dS  
dx ∫  ∫
------ = ρ – σ 1 I ( x, t ) exp  – σ 1 I ( x, t ) dt′ dt

(5.14)
0 0
which results in:
dS
------ = ρ ( 1 – exp ( – σ 1 S ) ) (5.15)
dx
What does Equation 5.15 imply about the laser deposition in such a target? At low pulse fluences, where
σ1 S « 1, the expression reduces to:
dS
------ = – εσ 1 S , (5.16)
dx
which, in light of Equation 5.8, is simply Beer’s law. At high fluences, where σ1 S » 1, Equation 5.16
simplifies to:
dS
------ = – ρ . (5.17)
dx
Equation 5.17 indicates that the absorption becomes saturated at high intensity. The maximum attenu-
ation rate of the incident photon flux per unit depth is simply equal to the density of chromophores in
the material.
Equation 5.15 can be integrated from the surface to an arbitrary depth x to find the residual number
of laser photons per area crossing x:
1 exp ( σ 1 S 0 ) – 1
S ( x ) = ----- ln 1 + ---------------------------------
- (5.18)
σ1 exp ( ρσ 1 x )
Returning to the “blow-off ” model, laser etching of the target should occur to a depth where S(x) falls
to the ablation threshold value, Sth, which again is simply the threshold fluence divided by the energy
per photon. This depth, d1, can be found from Equation 5.18:
( S 0 – S th ) 1 1 – exp ( – σ 1 S 0 )
- .
- + --- ln --------------------------------------
d 1 = --------------------- (5.19)
ρ α 1 – exp ( – σ 1 S th )
This expression is obviously quite different from Equation 5.7. It predicts the ablation behavior for a
material with saturable absorption at the laser wavelength.
We can test this theoretical description against the experimental results in Figures 5.5 and 5.6. If we
assume that the density of 248 nm chromophores in the polyimide target ρ = 4.5 × 1022 cm-3, then from
Equation 5.8, σ1 ≈ 5.5 × 10-18 cm2. Plugging these values into Equation 5.19 gives the predicted etch
depth–fluence relationship indicated by the dashed curve in Figure 5.5. Using the same Equation 5.5
constants in Equation 5.18 produces the calculated transmission behavior shown by the dashed curve in
Figure 5.6. In both cases, model agreement with experiment is quite good for laser pulse fluences below
~2 J/cm2. Above this fluence, other nonlinear processes, such as plasma formation, that are not accounted
for in this model, may play an important role.28 More important than the particulars of this mathematical
model is the simple fact that extending the “blow-off ” ablation theory to account for changes in target
absorption during the laser pulse greatly improves the theory’s accuracy in describing the etch–depth
fluence relationship.
How important is dynamic target absorption in tissue ablation? In the best-studied case, ArF laser
ablation of cornea, transmission of 193 nm laser radiation through corneal tissue has been observed to
decrease at high ArF laser pulse fluences. This transmission decrease is not due to increased reflection
or scattering of laser radiation from the ablation site,47,48 and hence represents enhanced laser absorption
in the tissue. The dominant chromophore in the cornea, the peptide bond in the structural collagen
molecules, is present in the tissue at a density of about 1021 cm-3.10 Using a value of 10,000 cm-1 for the
tissue absorption coefficient (roughly the geometric mean of reported values) and 50 mJ/cm2 for the
threshold laser fluence, the ablation threshold absorbed energy density is ~5000 J/cm3, or 5 x 1020 193
nm laser photons per cm3. Thus, as was the case for polyimide, at ablative laser fluences a significant
fraction of the target chromophores participate in the absorption process, and dynamic chromophore
changes are a plausible explanation for the observed change in tissue optics.
Laser-induced increases in absorption can be most readily accommodated in the model of Figure 5.7
by assuming that the excited chromophore state has a finite lifetime (relative to the laser pulse) and an
absorption cross-section larger than s1.43 A detailed numerical analysis here is not useful, because there
are substantial uncertainties in both the true value of α for this interaction and in the corneal etch depth
versus ArF laser fluence relationship.10 Suffice it to say that nonlinear absorption processes, e.g., chro-
mophore modification and plasma formation, undoubtedly do occur during tissue irradiation and do
impact on the ablation behavior.
5.3 Target Decomposition

Having described how the laser energy is deposited in the tissue, we must now consider the consequences
of that absorption. Figure 5.8 gives a simple schematic of the possible steps leading from radiation
absorption to ablation. The ultraviolet laser photons initially promote target chromophores to excited
electronic states. Some fraction of the absorbed energy may be reradiated, as occurs, for example, in
fluorescence. However, the vast majority of the pulse energy follows two pathways after the initial
absorption. An electronically excited chromophore may directly dissociate into two new species (“bond-
Absorption of Electronic Direct ''Bond-

UV Radiation Excitation Breaking''
Vibrational Denaturation/
Organic Relaxation Decomposition
Tissue
Component
Molecular Tissue
Collisions Ablation
Aqueous
Tissue
Component Expansion/
Heating
Vaporization
FIGURE 5.8 Simplified schematic of the energy pathways leading from laser irradiation to ejection of tissue
decomposition fragments (ablation). Laser radiation is primarily absorbed by the structural organic tissue compo-
nent. A combination of the weakening of this tissue scaffolding and generation of vapor phase decomposition
molecules drives the ablation event.
breaking”).5 This pathway may be particularly important at shorter ultraviolet wavelengths, where the
photon energy exceeds the bond strength of most organic bonds. Alternatively, the energy in the elec-
tronically excited chromophore can be redistributed to the rest of the macromolecule in the form of
vibrations (vibrational relaxation).49 This pathway is predominant for longer UV wavelengths, and likely
plays some role at shorter wavelengths as well.6–9
Large organic polymers such as proteins will contain numerous chromophores within each molecule.
Transfer of energy from multiple chromophores to the bulk macromolecule will drive it into pronounced
vibratory oscillations. If severe enough, these vibrations, in turn, cause denaturation, i.e., change in the
spatial folding of the biopolymer, and decomposition into smaller molecules. At the same time, because
the oscillating organic molecules in tissue are bathed in an aqueous environment, some thermal energy
transfer from the initial energy absorbers to surrounding water molecules also occurs. (Note that here
we are talking about heating on a nanoscopic scale, not thermal diffusion out of the laser absorbing
volume as described by Equation 5.1.) All of this occurs on a timescale faster than the nanosecond
duration of the laser pulse.49,50
We know that the target undergoes heating because we observe in it a pressure transient characteristic
of rapid thermal expansion (“Grüneisen stress,” as discussed below),50,51 as well as a bulk temperature
rise after the laser pulse.52–55 Less certain is the degree of heating that occurs during the laser pulse and
the contribution of such heating to the ablation event. Heating of the aqueous tissue component could
theoretically be both of large magnitude and very rapid, in part due to temperature related changes in
the ultraviolet absorption properties of water.22 Once initiated by heat transfer from organic molecules
early in the laser pulse, the warming aqueous component could then directly absorb a significant fraction
of the laser energy. If the tissue water within the laser-absorbing volume could be heated very quickly,
i.e., faster than it could physically expand, then the enormous resulting pressure (due to the relative
incompressibility of water) could drive the explosive ejection of material.56 Less rapid aqueous heating
could still contribute to the ablation event — not through laser absorption, but through steam formation.
These mechanisms are currently the subject of numerous laboratory investigations.
Denaturation and fragmentation of the organic tissue component greatly reduces the structural integ-
rity of the tissue.50,57 This structural weakening, combined with the internal pressure generated by gas
formation and thermal stresses, leads to the ejection of ablated material. There is good evidence that this
ejection begins during the nanosecond timescale UV pulse. Target stress wave measurements conducted
during photoablation of a variety of tissues50,51,58,59 as well as polyimide60 indicate the onset of material
removal during the laser pulse. The photoacoustic measurements in these studies indicated that, as the
incident fluence was increased, material ejection occurred progressively earlier during the laser pulse,
and that the process occurred via a rapid surface vaporization rather than a bulk explosion. In a separate
examination of XeCl laser polyimide ablation using an ultrafast imaging apparatus to look directly at
the target surface,61 etching of the polymer clearly occurred “layer by layer” over the course of the
irradiation. This “moving ablation front” was observable late in the irradiation at low ablative pulse
fluences, and early at high laser intensity, in general agreement with the photoacoustic studies.
Another measurable phenomenon that occurs during the laser irradiation is a marked fall in target
reflectivity. UV laser pulses specularly reflected from a host of synthetic and biologic materials under
ablation conditions exhibit a truncation in the latter portion of the temporal pulse profiles.62–64 In every
case studied to date, this reflected pulse “clipping” is first observable at incident laser fluences near the
ablation threshold and becomes increasingly more pronounced as the laser intensity rises. The reflectivity
decrease is transient,65 although it may persist for up to several hundred microseconds after the ultraviolet
laser pulse.66 Although the precise cause(s) of this phenomenon has not been completely established,
both debris plume attenuation effects and target index of refraction changes have been advanced as
possible mechanisms.62,65 Recent experimental measurements have, in fact, demonstrated refractive index
changes in polyimide during UV laser irradiation.67,68 Whatever the cause, the effect has been utilized
empirically to examine various aspects of the ablation process,62,69 and has been investigated as a diagnostic
feedback monitor for clinical photoablation procedures.64,70
If the heating occurs much faster than the material can move, significant Grüneisen stress builds up
in the laser-absorbing volume.71 To assess the magnitude of this phenomenon in a given situation, the
heating period, i.e., the laser pulse duration, must be compared with the expansion time of the heated
volume. In an fashion analogous to Equation 5.1, which defined a thermal relaxation time, an “acoustic
relaxation time,” τa, can be found by dividing the 1/e laser penetration depth by the speed of sound in
the material.72 The speed of sound in most tissues will be approximately the same as in pure water, ~
1500 m/s, so that:
1
τ a = ------------------------------------ . (5.20)
( 1500m ⁄ s ) ( α )
If the laser pulse is short compared with τa, the absorbing material is being heated much faster than it
can possibly expand, and the Grüneisen stress becomes important.
It is worth pointing out that, in Equation 5.20, the acoustic relaxation time is inversely proportional
to the absorption coefficient. Therefore, for 193 nm laser tissue ablation, where the absorption coefficient
is at least several thousand inverse centimeters, that is, on the order of a few nanoseconds. Because ArF
excimer laser pulses are typically 10–20 ns long, the Grüneisen stresses are probably not substantial in
these cases. For the 308 nm XeCl laser, tissue absorption coefficients may be more than two orders of
magnitude smaller, while the laser pulse duration usually does not exceed ~100 ns. Under those condi-
tions, the Grüneisen stress may become very important.52,72
5.4 The Ablation Plume

The most dramatic aspect of the ablation process is undoubtedly the visible explosion of debris from the
tissue surface, accompanied by an audible sharp snap. Principal features of this event are illustrated in
Figure 5.9. The ejection of ablated material causes generation of a recoil pressure wave that propagates
into the target,59,73,74 and which possibly could cause ancillary damage to the tissue. In addition, a shock
wave is typically formed in the ambient environment that moves away from the target ahead of the actual
ablation plume.73,75,76 The dynamics of the explosion are highly dependent on the environment. Confined
Ablation in Air Ablation in Liquid
incident laser pulse incident laser pulse
shock wave shock wave
ablated material
ablated material
tissue surface tissue surface
recoil momentum
recoil momentum
(a) (b)
FIGURE 5.9 Dynamics of the plume expansion following laser ablation. In air, the rush of material into the ambient
atmosphere creates a shock wave, as well as a recoil force pressing downward on the tissue target. Similar effects are
observed for ablation in a liquid environment, although the plume expansion, i.e., bubble formation, is constrained
by the fluid. This “tamping” leads to much greater recoil forces acting on the residual target.
plume expansion, e.g., expansion in a liquid such as blood or saline, is considerably retarded versus
expansion in air, and is accompanied by much greater recoil stresses in the remaining tissue.74,77
What are the products of ablative photodecomposition? The dominant constituent in a tissue ablation
plume is submicron diameter water droplets78 — not surprising, given the target composition. Mass
spectroscopy has been used to analyze the gaseous tissue ablation products. Corneal tissue ablation by
either 193 nm or 248 nm excimer laser produces a variety of organic gases, e.g., C2H2 and CH2NH2, in
addition to water vapor.79 Heavier molecular weight material, presumably macromolecular fragments,
are also observed. Except for the water, similar species are also observed in ablation plume from synthetic
polymers. Infrared spectroscopy and gas chromatography, as well as mass spectroscopy, have been used
to identify the principle gaseous products of polyimide photoablation: CO2, CO, H2O, and HCN, as well
as various light hydrocarbons (up to 4 carbon atoms in size).7,80 Larger molecules all the way up to visible
soot particles have also been identified.6,7,81
A consistent finding in these studies is that smaller molecular weight products are more prevalent as
higher laser pulse fluences are used. This trend can be explained by referring back to Figure 5.3. For two
laser pulses of different energy but the same wavelength, the average deposited energy in the ablated
volume is greater for the higher energy pulse. This extra energy fragments the plume debris into pro-
gressively smaller molecules and accelerates them to higher velocities.
There are also short-lived species that form early in the ablation event but do not persist long enough
to be detected by the above methods. For example, laser-induced-fluorescence measurements indicate
that the radicals C2 and CN form transiently during polyimide photodecomposition.6 Because of their
bioreactivity and potential deleterious impact on tissue, free radicals are particularly interesting tran-
sient ablation products. Electron paramagnetic resonance spectroscopy indicates that short-lived free
radicals are formed during both 193 nm irradiation of cornea,82 and during 308 nm irradiation of
cardiovascular tissue.83 In the former case, there is in vivo evidence that radical bioeffects do impact
the corneal healing response.84
One of the most straightforward and useful methods for studying the dynamics of material ejection
is laser-flash imaging.42 This technique is illustrated in Figure 5.10. A pulsed visible laser is used essentially
as a stroboscopic light source to generate an image of the ablation site on a camera or other imaging
device. By varying the delay between the firing of the ablating laser and the imaging pulse, it is possible
to capture clear images of the target explosion at discrete time intervals, and hence to assess ejection
variable
delay pulsed UV ablation laser
generator
target
pulsed visible laser imaging system
FIGURE 5.10 Ultrafast imaging technique used to study ablation plume expansion dynamics. By varying the delay
between the UV ablation laser and the visible imaging laser (essentially a stroboscopic source), the time course of
plume development can be explored. Motion artifacts due to the moving plume are reduced as the duration of the
imaging laser pulse is shortened.
velocities and particle sizes. The resolution of the image will improve as the visible laser pulse is shortened
because the blurring effect of motion is reduced. Successful imaging experiments of the photoablation
event have typically used visible imaging pulses of a few nanoseconds or less duration. Typically, the
optical axis of the imaging system is oriented parallel to the target surface to examine the orthogonal
development of the plume.
This technique was first used to study excimer laser ablation of corneal tissue in air at ArF laser
pulse fluences of 100-900 mJ/cm2 and KrF laser pulse fluences of 500 mJ/cm2.42 Using either excimer
device, the plume was first detectable 500 ns after onset of the 15 ns laser pulse. Images acquired at
slightly different delay times indicated that the cloud of particulate debris was initially traveling normal
to the tissue surface at transonic to supersonic velocities (≥ 340 m/s), but quickly slowed as it interacted
with the viscous air to roughly 100 m/s by 10 µs postirradiation. The plume velocity increased slightly
with increasing ArF laser pulse fluence, although the fastest plume expansion was observed during
KrF laser irradiation of the tissue. This was attributed to the greater ablation mass and larger particle
size at the longer ultraviolet laser wavelength (as discussed above) causing the plume to be less retarded
by air viscosity.
The major limitation of the imaging method is that it, of course, detects only solid particles. For
ultraviolet laser ablation, a significant fraction of the ablated material is in the form of low molecular
weight gases (as discussed below), which are essentially invisible as they expand into air. Other techniques
such as time-of-flight mass spectroscopy85,86 or probe-beam laser-induced fluorescence6,31 have been used
to examine the velocity of these ablation products. Results using these methods during ablation of
synthetic polymers show gas-phase products move away from the target surface at substantially supersonic
velocities (103-104 m/s).
In contrast, in an aqueous environment, the gaseous plume becomes a bubble, which can be examined
by ultrafast imaging. Such studies of bubble formation have been conducted during XeCl laser ablation
of arterial tissue in saline,87 as well as an absorbing hemoglobin solution.88 During the first ~100 µs after
a laser pulse, such bubbles expand with average velocities on the order of 20 m/s. As a bubble grows, the
temperature of the initially hot gas inside falls quickly, causing a rapid decrease in the driving pressure.
Eventually, after a few hundred microseconds, the compressive force of the surrounding fluid causes the
bubble to collapse. Interestingly, this collapse can be so violent in nature as to cause transient formation
of a second bubble (sometimes called “rebounding”).88
The duration of the material ejection in air has also been well studied using the imaging technique of
Figure 5.10. Images acquired during corneal ablation by the ArF excimer laser show debris leaving the
tissue surface more than 5 µs after the laser pulse.42 On a timescale of 10s to 100s of microseconds, the
ejected plume material is shown to disperse in the air above the target. Essentially similar results have
been reported for XeCl excimer laser irradiation of arterial tissue,89 and for KrF laser ablation of PMMA.76
As the ablated material begins to rush out of the target, it creates a blast or shock wave (a narrow layer
of extremely high pressure gradients moving at supersonic velocity), which then propagates into the
ambient environment ahead of the ablation plume. Such shock waves are also visible using the imaging
technique of Figure 5.10. These waves have been qualitatively observed to accompany ArF laser corneal
tissue ablation,75 and have been quantitatively studied during ablation of synthetic polymers.76,90 For KrF
laser ablation of polyimide, an initial blast wave velocity of 1200 m/s has been measured.90 As with the
plume, the shock wave velocity rapidly falls off with expansion into the air.
The recoil forces exerted on the remaining target by the ejected plume have been directly studied by
mounting thin (30–100 µm) slices of tissue on a piezoelectric transducer.50,59 Measurements made during
ArF laser corneal ablation indicate a transient pressure rise in the tissue of as much as 100 ATM due to
the stress wave. Target pressure wave formation has also been studied using an acousto-optical technique
and a synthetic model of corneal tissue: a thin sheet of polyimide floating on water.77 In that work,
irradiating the surface polymer with ablative XeCl excimer laser pulses was found to produce true shock
waves in the underlying water, with peak pressures calculated to exceed ~1000 ATM. The shock wave
amplitude was even larger (by a factor of 5–10) at any given fluence if the plume expansion was confined
by a second overlying water layer. Notable in all such studies was that the stress or shock wave amplitude
increased markedly with increasing laser pulse fluence.
These recoil forces in the target are not without biologic consequences. In one study of ArF laser, bone
ablation osteocyte destruction was observed more than 1 mm beneath the surface of the etch crater,91 in
all likelihood due to photoacoustic injury. Confining the ablation plume expansion, as occurs, for
example, during laser angioplasty in a fluid-filled blood vessel, has been demonstrated in several other
experiments to lead to greatly increased tissue damage surrounding the target area.88,89,92,93 As the mag-
nitude of the pressure wave in the target is a direct function of laser fluence, these findings suggest that
the most efficacious laser therapy will be achieved at moderate pulse fluences and, if possible, under
conditions where the ablation plume can expand freely.
It is often desirable to assess the recoil imparted to a tissue during ablation in a quantitative way. A
clear analysis of this topic has been presented previously by Dingus.75 The essential features of his discourse
are as follows. This discussion first considers the case of expansion of the ablation plume into free space,
then addresses the more complex situation of constrained or “tamped” vaporization.
The law of conservation of momentum indicates that the total momentum of the ejecta in the ablation
plume will be equal to the momentum delivered to the remaining target. If the ablation is assumed to
be uniform over an irradiated target of area A then:
∞
Mi vi
∑ i
----------- =
A ∫ p ( t ) dt , (5.21)
0
where Mi is the total mass of the plume debris traveling orthogonally from the target at velocity vi, and
p(t) is the pressure transient induced in the residual tissue. The integral of pressure over time is defined
as the specific impulse, noted here as I. Note that this expression treats the process as one-dimensional,
i.e., it assumes all ablated material is ejected perpendicular to the target surface. It is worth noting that
even completely isotropic ejection into 2π solid angle would reduce the imparted momentum by only
half. Furthermore, under most practical conditions, A is large enough so that the ejection is fairly
anisotropic and Equation 5.21 closely approximates the experimental result.
All the material is initially assumed to have approximately the same ejection velocity. If m0 and k are
defined respectively as the ablated mass per area and the kinetic energy per area (k = m0 v2 /2), then the
specific impulse is simply:
I = m0 v = 2m 0 k (5.22)
The kinetic energy per area in the ablation plume obviously cannot exceed the fluence F in the incident
laser pulse. This fact places an absolute upper limit on I, namely I max = 2m 0 F . However, in reality, I
is far less than Imax. The ratio of the specific impulse generated in the target to the incident laser pulse
fluence (I/F) is defined as the “impulse coupling coefficient.” Typical values of the impulse coupling
coefficient from experimental ablation measurements are 10-5 to 10-3 s m-1.
It is illustrative to apply these ideas to the experimental photoacoustic study of ArF excimer laser
cornea ablation mentioned earlier.59 In that work, laser-induced pressure waves were detected by a
piezoelectric transducer on the rear surface of 30 µm-thick corneal tissue slices. Irradiation of the sample
with ablative 16 ns laser pulses produced pressure pulses of approximately 30 ns duration (full width at
half maximum). At the highest experimental laser fluence, 570 mJ/cm2, the amplitude of the transducer
signal corresponded to peak pressure of about 100 ATM in the stress wave. In light of Equations 5.21
and 5.22, we can use these values to estimate the specific impulse generated in the cornea to be roughly
0.3 N s m–2 (since 1 ATM _ 105 n m-2). The impulse coupling coefficient in this case is therefore ~5 x
10–5 N s J–1. For comparison, the theoretical Imax under these conditions is an order of magnitude larger,
about 3.4 n s m-2, based on an ablation depth of ~1 µm @ 570 mJ/cm2 (see Figure 5.4) and assuming
the density of cornea to be around 1 gm/cm3.
I is always much less than Imax for at least three reasons (aside from any angular distribution in plume
particle velocities). First, not all the energy originally in the laser pulse ends up in the ablation plume.
Referring back to Figure 5.3, a finite part of the incident pulse is deposited deep in the tissue, beneath
the ablated region. (An additional small fraction of the pulse is reflected from the target surface.) Second,
not all the laser energy absorbed in the ablation volume is converted into the kinetic energy of the debris.
The energy density Eth is required just to photodecompose the target material. Third, the kinetic energy
in the plume is not uniformly distributed over the entire mass, as would be the case for maximal
momentum generation. Based on these considerations, Dingus has shown that the maximum value of
the impulse coupling coefficient is 0.5 to 0.6 times (Eth/µ)–1/2, where µ is the material density. In addition,
this optimal impulse coupling typically occurs when the laser pulse fluence is six to seven times the
ablation threshold.
Evaluation of these issues becomes much more complicated under tamped conditions (e.g., ablation
of a tissue in liquid).94 Analysis of conservation of momentum is no longer straightforward because there
are not two quickly separating masses (the plume and the residual target). The confining ambient medium
keeps the vaporized material in contact with the remaining bulk, and much of the energy originally
absorbed in the ablation region can be transferred back into the solid. In one study of metal ablation
under tamped versus untamped conditions, the kinetic energy coupled into the target mass was 1000
times greater with tamping than without.94 Tissue ablation in a fluid, as occurs during laser angioplasty,
is a practical example of at least partially tamped. In general, such enhanced coupling is lessened if the
tamping medium has a high thermal conductivity or the ablation plume contains condensable vapors
that are likely to precipitate out of the plume on striking the cold tamping medium. Both these qualities
facilitate rapid transfer of energy from the plume into the ambient environment rather than into the tissue.
The shock wave generation accompanying UV-laser photoablation is a complex nonlinear process.
Nevertheless, it is important to have some understanding of such waves because of their potential to
cause damage in delicate tissues. A simple yet instructive conceptual picture of this phenomenon has
been presented in Ref. 95, and the essential features of that description are shown in Figure 5.11. Consider
a laser pulse of cross-sectional area A striking a target at time t = 0 and generating an ablation plume
that then rushes out into the fluid ambient environment at a velocity u. The environment initially has a
uniform density, µ0, as indicated in the lower left hand of the figure. As the plume pushes outward, a
leading-edge “bow wave” of swept up fluid is created. If the plume velocity is high, i.e., at least on the
order of the ambient sound velocity, then the stress in the bow wave becomes large.
Important to note here is that, at high compressive stress, the wave propagation velocity is no longer
constant (the characteristic sound speed), and generally increases with increasing pressure. Therefore,
as the amplitude of the bow wave increases, the region of peak pressure begins to move faster than
the rest of the wave. This leads to a marked sharpening of the leading edge of the wave into a very
thin layer with a nearly discontinuous jump in pressure (hence the damage potential), and moving at
supersonic velocities. The developed shock wave is shown in the right half of Figure 5.11 at time t
after the laser pulse. For simplicity, the shock front is modeled as a discontinuous layer of uniform
time = 0 time = t
target ambient medium
plume
area = A shock front
laser pulse u U
µ1
density
density
µ0
? µ0
distance ut Ut
FIGURE 5.11 Theoretical model of shockwave propagation into an ambient environment on density µ0 following
laser target ablation. The shock front is modeled as a thin layer of density µ1 propagating at a velocity U. The expanding
ablation plume, of unknown density, propagates at an average velocity u behind the shock front (see text).
density µ1. The leading edge of the discontinuity propagates into the undisturbed ambient region at
a velocity U.
Because of conservation of mass, the total material swept ahead of the plume must be contained in
the shock region:
µ 1 A ( Ut – ut ) = µ 0 AUt , (5.23)
meaning that the lightly shaded regions in the two graphs of Figure 5.11 have the same area. This mass
acquires a momentum of µ0 A Utu. Using Equation 5.21, we can relate the momentum per area in the
compressed layer to the specific impulse associated with the driving pressure. If p0 and p1 denote
respectively the pressures in the undisturbed ambient fluid and the shock region, then the net outward
pressure acting on the shock front is p1–p2, and:
µ 0 Utu = ( p 1 – p 0 )t . (5.24)
Because p1 » p0 under shock wave conditions, the ambient pressure is inconsequential,96 and Equation
5.24 simplifies to:
µ 0 Uu = p 1 . (5.25)
Equation 5.25 is known as a jump condition, and is a very useful tool for calculating shock wave peak
pressure based on velocity measurements.73,96 Note, however, that two velocities, those of the shock front
and the driving particles, are required to perform the calculation. While the former is relatively straight-
forward to measure, the latter can be extremely difficult to obtain. Fortunately, the two speeds are related
by the equation of state. To a first-order approximation:
U = a + bu , (5.26)
where a and b are empirically determined constants.96 For water, a = 1483 m/s (the speed of sound) and
b = 2.07.97
Shortly after the laser pulse, the shock wave separates from the ablation plume, and, from that instant
onward, the momentum in the shock region remains constant. This does not mean, however, that the
shock wave persists indefinitely. As soon as it is no longer being driven by the plume, both the pressure
and velocity of the shock fall precipitously. This is, in part, due to a geometric effect: as the shock moves
away from the ablation site, it evolves from a nearly plane wave into a spherical wave,76,90 and the intensity
of such a wave must have an inverse dependence on radius. In addition, shock waves heat the medium
they are propagating through,95 and this heating takes energy out of the wave. Such processes eventually
reduce shock waves to conventional pressure waves.
5.5 Repetitive Irradiation

Medical ablation procedures employ a train of many laser pulses to achieve appreciable tissue removal.
The quality of an ablation crater formed by repetitive irradiation, i.e., the depth, topography, and extent
of tissue damage, depends on specific laser parameters, tissue characteristics, and ambient conditions, as
described in the sections above. In general, it is assumed that each laser irradiation in a train of identical-
fluence pulses removes a consistent amount of tissue, so that the crater depth increases linearly with the
number of irradiations.
For certain synthetic polymers, such as PMMA, the relationship between crater depth and number of
laser pulses is complicated by a phenomenon termed “incubation,” particularly so for pulse fluences just
above the ablation threshold. Incubation is a process whereby multiple irradiations are required to initiate
etching at modest laser intensities. This effect has been extensively studied44,98–100 and has been shown to
be due to progressive photochemical alterations within the irradiated material (i.e., fragmentation of
large polymer molecules, formation of new UV absorbers, and accumulation of trapped gases). As for
tissue targets, incubation has specifically been shown not to occur during 193 nm corneal ablation,69 but
may play some role in vascular tissue irradiation at 308 nm, where irradiation-induced long-lived changes
in target optics have been observed.55
Another process, not yet documented for ultraviolet laser ablation of tissue but known to occur
with synthetic polymers, is “radiation hardening,” where material removal occurs only for initial pulses
in a train of irradiations. This phenomenon has been extensively observed for polyimide ablation, and,
like incubation, is most pronounced for pulse fluences in the vicinity of the ablation threshold. There
are at least two causes for radiation hardening. First, weakly ablative polyimide irradiation ejects low
molecular weight gases and leaves behind a surface layer of carbon crystallites.101 This carbon layer is
much more resistant to ablation than the original polymer. Second, low fluence ablation of polyimide
leads to a progressive roughening of the crater floor through the formation of randomly scattered
conical features,102–104 as well as periodic ripple-like surface waves.105 Roughening of the surface
increases the irradiation area, and hence decreases the effective fluence striking the target.
For tissue ablation, one established important consideration is the maximum repetition rate that can
safely be used in a clinical procedure. Referring once again to Figures 5.1 and 5.3, some fraction of the
incident laser energy for each pulse is deposited in the intact tissue below the ablation depth. This energy
is primarily converted to heat. If the subsequent laser pulse arrives before sufficient thermal diffusion
into the surrounding bulk tissue occurs (Equation 5.1), a pronounced localized temperature rise, and
hence thermal damage, can occur. In one study of ArF laser corneal ablation using a microthermocouple
technique,53 a temperature rise of 20 °C was obtained during excised tissue irradiation at 30 Hz with 360
mJ/cm2 laser pulses. Not surprisingly, the heating was found to increase with either increasing laser pulse
frequency or pulse fluence. A major conclusion of that study was that the pulse repetition rate should
be kept below 63 Hz for corneal sculpting procedures for conventional ArF laser fluences (below ~200
mJ/cm2 per pulse).
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6
Tissue Diagnostics
Using Lasers
6.1 Introduction ....................................................................... 135

6.2 Light Interaction with Tissue ............................................ 136
Molecular Absorption of Laser Light • Laser-Induced Tissue
Heating • Laser-Induced Photochemistry • Laser-Induced
Fluorescence • Elastic Scattering • Raman Scattering
6.3 Spectroscopic Diagnostics of Malignant Tumors ............ 141
Basic Considerations • Instrumentation for Point Monitoring
of Laser-Induced Fluorescence • Point Monitoring of Cancer
using Laser-Induced Fluorescence • Equipment for Imaging •
Multi-Color Fluorescence Imaging of Tumors
6.4 Spectroscopic Diagnostics of Atherosclerotic Plaque...... 151
Basic Considerations • Plaque Discrimination Using Laser-
Induced Fluorescence • Time-Resolved Monitoring • Raman
Spectroscopy for Tissue Diagnostics • Tissue Reflectance
Monitoring
6.5 Light Scattering and Tissue Transillumination................ 157
Doppler-Flow Monitoring • Transillumination Imaging •
Interpretation of Time-Resolved Tissue Recordings
6.6 Outlook ............................................................................... 163
Sune Svanberg Acknowledgments......................................................................... 164
Lund Institute of Technology References ...................................................................................... 165
6.1 Introduction
While therapeutic aspects of lasers in medicine have been very dominant, tissue diagnostic techniques
using laser spectroscopic techniques are now emerging strongly. Applications include endoscopic cancer
detection using fluorescence, plaque monitoring in the circulatory system including coronary arteries,
and tissue transillumination for optical mammography. Several factors contribute to the increasing
interest in laser tissue diagnostics:
• Optical monitoring is non-intrusive in nature.
• Non-ionizing radiation is employed.
• Real-time data representation is possible.
• Spectroscopy allows molecular specificity in analysis.
• Point monitoring or imaging capability can be employed.
• Integration of laser diagnostics and therapy is possible.
0-8493-1146-2/02/$0.00+$1.50
Sometimes the term “optical biopsy,” which also gives the flavor of the attractive features of the new
emerging methods, is used. In comparison with well established diagnostic modalities such as x-ray
radiography, NMR imaging, ultrasound imaging and positron emission tomography,1 the optical spec-
troscopic techniques are in an early stage of development. However, through extensive work at several
research centers, the optical techniques are developing swiftly, and clinically useful equipment is now
becoming available.
The organization of the present chapter is as follows: Light interaction with tissue, forming the basis
for optical tissue diagnosis, is reviewed in Section 6.2. In a following section, malignant-tumor diagnostics
based on laser-induced fluorescence is treated, where point monitoring and multi-spectral imaging, as
well as time-resolved aspects, are included. Monitoring of the interior of vessel walls and of the heart
muscle is treated in Section 6.4, with a discussion of the possibilities of integration of diagnostics with
a treatment modality. Finally, light scattering in tissue transillumination is treated in Section 6.5, which
includes Doppler perfusion monitoring, optical dosimetry for photodynamic therapy and the detection
of deep-lying malignant breast tumors using time-gated viewing. An outlook for the future concludes
the chapter.
6.2 Light Interaction with Tissue

Optical diagnostics for medical purposes is based on the interaction of light with tissue. The interactions
can be resonant or non-resonant in nature:
• Resonant
• Molecular absorption of laser light
• Laser-induced tissue heating
• Laser-induced photochemistry
• Laser-induced fluorescence
• Non-Resonant
• Elastic scattering
• Raman scattering
Light–matter interaction mechanisms, spectroscopic instrumentation and measurement techniques
are treated in text books on atomic and molecular spectroscopy.2 Here, we will now discuss the different
modes of interaction between light and tissue.
6.2.1 Molecular Absorption of Laser Light

When tissue is irradiated by laser light, a small fraction of the light is reflected, but most of it penetrates
into the tissue, where it is either absorbed of scattered by the molecules. In certain wavelength regions,
absorption strongly dominates over scattering. The stronger the absorption, the smaller the penetration
depth into the tissue will be.
A schematic diagram of the absorption of some important biological constituents and chromophores,
such as water, proteins, nucleic acids, hemoglobin and melanin is shown in Figure 6.1.3 Here also, the
emission wavelengths of some important medical lasers are included. As can be seen, water has two
regions of strong absorption, one in the deep ultraviolet (UV) region and one in the infrared (IR) region.
The absorption maximum in the case of the aromatic rings of proteins and the nucleic acids is in the
UV region between 260 and 280 nm. Thus, in the UV region, the laser light is highly absorbed by the
water and proteins in the tissue, resulting in a poor light penetration into the tissue. The same is true in
the IR region, starting at the second water absorption region for water at about 1.3 µm. Hemoglobin in
the blood absorbs light in a broad wavelength region up to red light (about 600 nm), with a pronounced
absorption maximum at about 400 nm and additional weaker peaks in the green/yellow region. Above
600 nm, the absorption of blood is weak. Melanin, the most important chromophore in the epidermis,
Tissue Diagnostics Using Lasers 137
ENERGY E(av)
102 10 8 6 4 3 2 1 0.8 0.6 0.4 0.2 0.1
MOLAR EXTINCTION COEFFICIENT e(1031/mol-cm)

104
10-17
H2O
106
CROSS SECTION (cm2)

DOPA- 10-18
Melanin
HbO2
108
Adenine 10-19
H2O
10
10-20
Copper Argon
1 HPD
Nd-YAG (4 w)
Nd-YAG (4 w)
HbO2 10-21
Dye
Nd-YAG
Copper
Ga-As
He-Ne
Argon
10-1
XeCl
Gold
CO2
Krf
Arf
10-22
10-2
100 200 300 600 700 1000 2000 4000 10000
WAVELENGTH l(nm)
FIGURE 6.1 Absorption properties of biological molecules: water, hemoglobin, melanin, hematoporphyrin deriv-
ative (HPD) and adenine (From Ref. 3).
absorbs light in a region from UV to near IR. Between 600 nm and 1.3 µm, the attenuation coefficients
of the tissue molecules are comparatively small, resulting in a wavelength region with favorable light
penetration properties, with increasing penetration toward the IR region. Considering the total tissue
absorption, it can be noted that the human body is as transparent as can be at about 1 µm.
The absorptive properties of a surface can also be monitored indirectly in reflectance. This is a well-
known phenomenon that forms the basis for the human perception of colors. For example, the red color
of blood can be understood from the absorption curve given in Figure 6.1, where all colors below 600
nm in the impinging white light are strongly absorbed by the hemoglobin molecules. Red light, however,
is not absorbed, but can be scattered, reaching the eye of the observer. In practice, reflectance spectroscopy
performed by the doctor’s eye and coupled to the processing of an experienced brain is a most important
means of assessing human tissue status. The technique can be refined by using a spectrometer, which
also gives access to the IR region, where characteristic absorption bands due to molecular vibrational
transitions modify the impinging radiation. This technique can basically yield the same type of infor-
mation as Raman spectroscopy, discussed below. Analysis of reflected light forms the basis for global
earth-resource monitoring using passive satellite sensors.4
6.2.2 Laser-Induced Tissue Heating

Laser-induced tissue heating occurs through the conversion of electromagnetic energy into thermal
energy. This photothermal process takes place when biomolecules have absorbed light quanta to arrive
in an excited state and return nonradiatively to lower excited levels, transferring their energy to the tissue.
In the thermal mode of tissue interaction the choice of wavelength, as well as the tissue type, determine
the light penetration depth. Heating leads to protein coagulation (60 °C), body fluid boiling (100 °C)
and tissue carbonization (200 °C) that can be used for hemostasis and tissue surgery. This is a major
application of lasers in medicine, having much impact in opthtalmology, dermatology and general
surgery. The carbon dioxide laser (10.6 µm) has a very low tissue penetration due to water absorption,
as has the argon-ion laser (488, 515 nm), due to hemoglobin. If a dye laser is tuned to 580 nm, it can
be made to be absorbed quite selectively by the hemoglobin in small vessels (see Figure 6.1). Thus,
hemangioma, such as port-wine stains, can be efficiently treated while sparing surrounding, normal
tissue. Maximum penetration occurs for the Nd:YAG laser (1.06 µm), which induces wide coagulation
zones around surgical cuts. The various clinical applications of lasers operating in the thermal mode are
discussed in many chapters of this book. For the purpose of tissue diagnostics using laser techniques,
thermal effects should be avoided by keeping the laser fluence sufficiently low. We include this brief
discussion of laser thermal interaction only to clarify the limitations of optical tissue diagnostics.
For UV wavelengths, the penetration depth of laser light is only of the order of 1–100 µm. Such
radiation can be used to induce fluorescence in the biomolecules, which provides an important means
of tissue diagnostics. However, high incident fluences of such radiation in short pulses leads to explosive
tissue ablation, and must thus be avoided in all diagnostic work. Laser tissue ablation, on the other hand,
has important medical applications for myopia correction by corneal reshaping and as a modality for
clearing occluded vessels using transluminal optical fibers, as discussed in Chapters 85,6and 9.
6.2.3 Laser-Induced Photochemistry

Optically excited molecules can, instead of releasing their excess energy as heat, transfer their energy to
other molecules, thus inducing chemical reactions of therapeutic interest. Optical treatment of psoriasis
patients or newborn infants with an excess of bilirubin are well-known examples of medical photochem-
istry. Photodynamic tumor therapy (PDT) using tumor-seeking agents is a further example of special
interest in our context. The most commonly used tumor sensitizer is hematoporphyrin derivative (HPD),
which is commercially available under the trade name of Photofrin.
The absorption curve of HPD is included in Figure 6.1. When injected intravenously, such molecules
are selectively retained to a higher degree in malignant tumor tissue than in other surrounding tissue. It
can be noted in the figure that the absorption maximum of HPD is around 400 nm, similar to hemoglobin
in the blood, but also, that HPD exhibits four more absorbing peaks, with the last one at 630 nm, where
blood absorption is strongly reduced. Therefore, to penetrate deeper into the tumor tissue, laser light of
630 nm instead of 400 nm is used.
A schematic diagram showing the energy levels of the HPD molecule with absorption and fluorescence
emission wavelengths is shown in Figure 6.2.7 Following light irradiation, HPD molecules become excited.
The molecules can then be de-excited following two main pathways. In the first, the molecules are
transferred nonradiatively to the bottom of the first excited band, from whence a radiative decay follows
with the emission of a dual-peaked fluorescent light distribution in the red spectral region. This emission
can be used for the diagnostics of malignant tumors, because the characteristic red fluorescence follows
only from irradiated tissue that has retained the HPD molecules. This diagnostic aspect will be discussed
extensively in the next subsection.
In a second de-excitation pathway, the molecules are transferred into the triplet state. From here, the
excitation energy can be transferred to ground-state tissue oxygen molecules that are excited into their
singlet state. Singlet state oxygen is known to be an extremely toxic agent for living cells. As the HPD in
the cells mediates this energy transfer, which results in singlet-oxygen formation, the cells are transformed
from viable malignant cells to nonviable necrotic cells. This is the essence of photodynamic therapy,
which is now being pursued on an experimental basis at many centers throughout the world. The
therapeutic aspect, as well as the diagnostic, are schematically illustrated in Figure 6.3.7 Reviews of clinical
PDT have been given in Refs. 8 and 9. The field is further treated in Chapter 10 of this volume.10
6.2.4 Laser-Induced Fluorescence

Laser-excited molecules can return to the ground state by emitting fluorescent light. Fluorescence spectros-
copy is treated in detail in Refs. 11 and 12. The fluorescence always occurs for longer wavelengths (Stokes
shift). For large biomolecules, the fluorescence normally exhibits a rather structureless intensity distribution
as a function of wavelength, reflecting the distribution of substates in the ground electronic level. However,
by choosing the excitation wavelength properly and accurately analyzing the fluorescent light distribution,
it is frequently possible to obtain diagnostic information for the tissue molecules. Using short-pulse exci-
HPD O2
S2
S1
405 nm
500 nm
530 nm
570 nm
630 nm
690 nm
ENERGY
TRANSFER
T1
a1∆g
1270 nm
S0 x 3Σg
SINGLET OXYGEN
600 700 nm
INDUCES TISSUE
HPD FLUORESCENCE NECROSIS
FIGURE 6.2 Energy level diagram for the HPD molecule and illustration of HPD fluorescence emission and energy
transfer to oxygen molecules.7
tation, it is also possible to measure the decay time for the fluorescent light, yielding additional information.
Many different chromophores may contribute to the tissue signal: endogenous molecules such as NADH,
NAD+, collagen and elastin, giving rise to so-called auto-fluorescence, and additional tumor-seeking agents
such as hematoporphyrin derivative, phtalocyanines and chlorins, all featuring sharp, characteristic signals
in the red and near-IR region. Examples of fluorescence spectra for different pure tissue substances and of
tumor-seeking agents injected into the same type of tumor-bearing rats are shown in Figure 6.4.13,14
6.2.5 Elastic Scattering

Light scattering in tissue is important for red and near-IR light, which is not strongly absorbed by the
tissue constituents. The scattering is normally elastic, i.e., no change in wavelength occurs. If absorption
is low, many scattering processes may occur in a more or less random way before the photon is ultimately
absorbed or escapes from the tissue. The fate of an individual photon is determined in a complex interplay
between scattering and absorption. For certain geometries, the process can be described by solving the
diffusion equation, specifying values for the absorption and scattering coefficients µa and µs, respectively.
Of special interest is the study of transillumination through a thick tissue sample (few cm). Many of the
photons have been scattered many times, resulting in effective pathlengths of tens of centimeters. If large
computer capacity is available, the photon propagation can be simulated using Monte Carlo techniques,
where photon trajectories are followed for a large number of events to build up an intensity distribution.
In such calculations, the mean cosine value between the incoming and scattered photon directions of
propagation is also included.15–19
The presence of scattering makes tissue absorption measurements much more involved, as discussed
above. The pathlength in tissue that has been traversed cannot be assessed in steady-state measurements,
making a direct use of the Beer-Lambert absorption relation impossible. Time-resolved monitoring of
picosecond pulses, on the other hand, allow well-defined measurements, which are of particular interest
for measuring tissue oxygenation, using absorptive spectral features in the red/near-IR region as shown for
oxygenated hemoglobin in Figure 6.1. De-oxygenated hemoglobin has a different spectral structure.
Inject Wait
HPD 2-3 HPD is selectively
3 mg/kg days retained in tumors
Autofluorescence
HPD
UV signal
Laser
400 600 nm
630 HPD O2
nm
Singlet
Oxidation
Triplet
FIGURE 6.3 Schematic diagram of tumor marking by sensitizing molecules and the subsequent use for fluorescence
diagnosis and photodynamic therapy.7
If light is scattered against a moving particle, a very small frequency shift proportional to the in-line
velocity occurs due to the Doppler effect. The only important moving particles in the body are the red
blood cells. In the superficial capillary vascular bed accessible for non-intrusive light scattering measure-
ments, the flow velocities are low, typically 1 mm/s. For the helium-neon laser wavelength (633 nm) this
corresponds to a frequency shift of only about 3 kHz out of about 5 × 1014 Hz. Measurements of such
small shifts can be accomplished by a heterodyne technique, where the beat frequency between the
frequency-shifted light and the unshifted light scattered from the fixed cell structures is observed. Since
the scattering geometry is undefined, an average of different flow velocities and flow directions is obtained,
yielding an effective signal that is related to the superficial blood perfusion of the investigated tissue.
Laser Doppler techniques for tissue perfusion measurements are treated in Refs. 20 and 21.
6.2.6. Raman Scattering

Raman scattering is inelastic, leading to scattered photons of strongly changed wavelength. It may be
seen as an excitation to a virtual (non-resonant) state and an immediate transfer from this state to a
lower state of different energy from that of the initial state. In cases of medical interest, the lower state
splittings are due to vibrational energy. The resulting shifts are characteristic for vibrations in specific
molecular groups. Although Raman scattering provides a much higher molecular specificity than laser-
induced fluorescence, there is a sensitivity problem, because Raman scattering is many orders of magni-
tude weaker than laser-induced fluorescence. Thus, unless proper precautions are taken, the signals tend
to be swamped by the fluorescence signals.
COLLAGEN NADH CAROTHENE

ELASTIN a)
FLUORESCENCE INTENSITY
Trp
x1/2
350 400 450 500 550 600 650 700

WAVELENGTH (nm)
b)
FLUORESCENCE INT. (arb. units)
HP
DHE
PHE
BPD-MA
TSPc
450 550 650 750

WAVELENGTH (nm)
FIGURE 6.4 Fluorescence spectra for (a) pure tissue constituent molecules and (b) a number of tumor sensitizing
agents, accumulated in the same type of experimental rat tumor. (From Refs 13,14). Trp: tryptophan, HP: hematopor-
phyrin, DHE: dihematoportphyrin ether (or Photofrin), PHE: polyhematoporphyrin ester, BPD-MA: bensoporphyrin
derivative — mono acid, TSPc: tetra sulphonated phtalocyanine.
6.3 Spectroscopic Diagnostics of Malignant Tumors
6.3.1 Basic Considerations

This section will focus on tumor diagnostics using laser-induced fluorescence, and will comment only
briefly on Raman spectroscopy, which is presently less developed. At first glance, it might seem a
formidable task to derive any information from tissue fluorescence, considering the many types of
complex organic molecules that make up living tissue. We will first present typical tissue spectra in
Figure 6.522,23 and discuss the type of information it contains. The sample chosen is a rat tumor in a
muscle environment,24 and we show spectra for tumor as well as for surrounding normal tissue and for
two excitation wavelengths, 337 and 405 nm. The rat was injected with a dose of hematoporphyrin
derivative 2 days earlier. For both excitation wavelengths, a broad intensity distribution extending from
the blue to the near IR region is obtained. In the measurements, the strong, elastically scattered light
from the primary laser excitation light is suppressed by a suitable filter, completely blocking shorter
wavelengths but letting all longer wavelengths through basically unattenuated.
Fluorescence Intensity
TUMOR MUSCLE
l = 337 nm l = 337 nm
exc exc
500 600 700 nm 500 600 700 nm

Wavelength Wavelength
TUMOR MUSCLE
l = 405 nm l = 405 nm
exc exc
500 600 700 nm 500 600 700 nm

Wavelength Wavelength
FIGURE 6.5 Laser-induced fluorescence spectra for an experimental tumor (a human colon adenocarcinoma
inoculated in rat muscle), and normal surrounding tissue. The rat had been injected with a dose of hematoporphyrin
derivative about 2 days before the investigation. Two excitation wavelengths, 337 and 405 nm, are chosen to illustrate
the influence of the wavelength choice (From Ref. 23).
In the tumor spectra, we can see the characteristic dual-peaked fluorescence signal from HPD in the
red wavelength region (see Figures 6.2 and 6.4). This signal is less prominent in the normal tissue due
to the selective retention of the drug in tumor tissue. The signal in the blue-green region is called the
autofluorescence, as it is due to the natural chromophores in the tissue. This signal peaks at about 470
nm. The blue-green fluorescence is much stronger and the red HPD signal much weaker when 337 nm
is chosen as the excitation wavelength. This can be understood from the absorption curves in Figure 6.1.
405 nm corresponds to the absorption maximum (the Soret band) of the HPD molecules, and excitation
here is more efficient than at 337 nm. Many chromophores contribute to the signal in the blue-green
wavelength region (examples are given in Figure 6.4a). The absorption for most of these molecules
increases for shorter wavelengths. An interesting observation relating to Figure 6.5 is that the intensity
of the blue-green signal is reduced in tumor tissue as compared with normal tissue. This was noted
already in 198425 and seems to be due primarily to a transformation of strongly fluorescing NADH in
normal tissue to NAD+ with a much weaker fluorescence.26–28 These observations form the basis for
efficient tumor detection and demarcation using laser-induced fluorescence after injection of a tumor-
seeking drug: the red fluorescence increases and the blue-green fluorescence decreases in a tumor. Both
effects can be utilized in a convenient way by dividing the red signal intensity by the blue intensity.25,29
This is illustrated in Figure 6.630 for the case of a scan through a tumor inoculated in a rat brain. The
rat had been injected with Photofrin (1 mg/kg bodyweight) 24 hours before the animal was sacrificed
and the fluorescence investigation was performed. Typical spectra for normal and tumor regions are
included in the figure, with the red intensity at 630 nm denoted by A′ and the blue intensity at 470
denoted by B. The background-free HPD signal at 630 nm is denoted by A. In the scan, the red increase
and the blue decrease in the tumor are very evident. Contrast enhancement is obtained by forming the
ratio A–B. The use of such a dimensionless ratio also has other important advantages, particularly in
practical clinical work, as dimensionless quantities are immune to:
• Distance changes between tissue and measurement equipment
FUNCTION VALUE
arb. units
INTENSITY INTENSITY
rel. units rel. units
TUMOR NORMAL
80
10 TISSUE BRAIN
TISSUE
8 60
A'
6 40
A'
4 B A
20
2
A A/B
400 500 600 700 400 500 600 700
WAVELENGTH / nm WAVELENGTH / nm
-2 0 2 4 6
DISTANCE / mm
brain tumor brain
FIGURE 6.6 Fluorescence data obtained in a scan across an experimental glioma (TCVC) in a rat injected with 1
mg/kg b.w. Photofrin 24 hours earlier. Laser-induced fluorescence spectra of tumor tissue and surrounding normal
brain parenchyma are also shown. The background-free HPD-related signal is denoted A, while the total signal at
630 nm is denoted i.e., A′ and the autofluorescence B. The dimensionless ratio A/B shows a strong contrast enhance-
ment (From Ref. 30).
• Variations in angle of incidence of radiation on tissue

• Fluctuations in illumination source and detection system efficiency
The ratio signal is basically sensitive only to the intrinsic properties of the tissue. In particular, if the
drug-related signal is weak and sitting on a substantial slope of autofluorescence intensity, to retain
contrast, it is important to use the background-free signal intensity.
6.3.2 Instrumentation for Point Monitoring of Laser-Induced Fluorescence

This section will present equipment suitable for fluorescence studies of tissue. A sealed-off nitrogen laser,
emitting at 337 nm, is a very convenient excitation source. This laser typically generates 3 ns-long pulses
at 10 Hz, and can also be employed to pump a compact dye laser, converting the pump radiation to a
longer, selectable wavelength. This is the excitation source used in the recording of the data in Figures
6.5 and 6.6. Fluorescence is best analyzed with an optical multichannel analyzer system consisting of a
spectrometer equipped with an intensified diode-array detector in the focal plane. For each exciting laser
pulse, the whole fluorescence spectrum is captured and can be directly displayed on a monitor and stored
in a computer. The image intensifier, providing amplification of the weak light signals, is gated synchro-
nously with the laser pulses. In this way, background radiation due to the light from operating lamps
can be suppressed in the recordings.
In laboratory investigations, the excitation light can be directed to the sample by means of mirrors
and the resulting fluorescent light can be collected by an optical set-up of mirrors and lenses. The
fluorescence thus captured can be directed into the entrance slit of the spectrometer. However, for clinical
measurements, a more flexible fluorosensor is needed. Such instrumentation is shown in Figure 6.7.31
The excitation laser light from the laser is focused into an optical fiber that can be sterilized when used
in surgical procedures or used through the biopsy channel of an endoscope. The fluorescence is captured
through the same fiber separated from the excitation light by a dichroic mirror. The fluorescent light is
ELECTRICAL SIGNALS TO
AND FROM OMA MAINFRAME
DETECTOR
NITROGEN
LASER
POLYCHROMATOR
LENS
MIRROR FILTER
FLIP-IN
MIRROR
CUT-OFF
FILTER DYE LASER
LENS LENS FILTER

OPTICAL FIBER DICHROIC
MIRROR LENS FLIP-IN MIRROR MIRROR
MIRROR
LIGHT TO AND
FROM TARGET
FIGURE 6.7 Lay-out of a clinical fluorescence diagnostic system (From Ref. 31).
focused onto the slit of a multichannel analyzer system. The fluorosensor is constructed as a mobile
system and can easily be transported between different clinics. Two systems of this construction are used
for fluorescence studies at various clinics at the Lund University Medical Laser Center. Most of the
examples of fluorescence spectra shown in this chapter have been recorded with this type of equipment.
Interference filters can also be used to select the interesting wavelengths for fluorescence signal detec-
tion. The fluorescent light can then be divided by dichroic beamsplitters and directed toward individual
photomultipliers. One detection channel can be centered at the 630 nm peak, while another one is set
at 470 nm. An additional channel can be centered at 600 nm to provide a signal for background
subtraction. Gated integrators are used to capture the signal during a brief time interval, followed by
signal processing to provide the desired ratio signal.
It is also possible to use a mercury lamp as an excitation source instead of a laser. Such a system is
described in Ref. 32, and was further developed and tested as described in Ref. 33. A chopper wheel
equipped with interference filters analyzes the fluorescent light beam and filters out two excitation
wavelengths, i.e., 365 and 405 nm. The filtered light is reflected by a dichroic beam splitter and is focused
into the investigation fiber. The fluorescent light comes back through the fiber. Here, interference filters
sequentially select the useful wavelengths, i.e., 470, 600 and 630 nm for both excitation wavelengths in
cases of tumor diagnostics utilizing HPD sensitization. The light is recorded by a photomultiplier tube,
and the signal is electronically integrated during the passage of every filter. The result is digitized and
fed to a computer. In this way, three fluorescence wavelengths can be investigated at two excitation
wavelengths for every tissue spot under study.
6.3.3 Point Monitoring of Cancer using Laser-Induced Fluorescence

This section will give some examples of point monitoring of fluorescence related to malignant tumor
detection. Data for superficial tumors in humans are shown in Figure 6.8.34,35 In the left part of the figure,
fluorescence spectra and evaluated data from a scan are shown for a basal cell cancer in a patient who
was injected by 2 mg/kg body weight of Photofrin 3 days before the fluorescence measurements. In the
right part of the figure, data for a breast carcinoma metastasis are shown in a similar way. This patient
was studied 1 day after the same dose of Photofrin. A good demarcation of the tumor is obtained for
both tumors, particularly in the A/B signal.
FUNCTION
VALUE
FUNCTION VALUE
FLUORESCENCE FLUORESCENCE
arb. units
INTENSITY INTENSITY B
TUMOR TUMOR
A B A
B
500 600 700 B
SKIN 500 600 700
SKIN
A/B A/B
500 600 700 A A
WAVELENGTH/nm -6 -4 -2 0 2 4 distance/cm 500 600 700 20 10 0 10 20
skin tumor skin WAVELENGTH/nm DISTANCE/mm
FIGURE 6.8 Fluorescence spectra and evaluated data from two different human malignant tumors and normal
surrounding skin. To the left two fluorescence spectra and evaluated data from a scan across a basalioma 3 days after
Photofrin incjection (2 mg/kg body weight). To the right two corresponding spectra and data from a scan across a
breast carcinoma metastasis 1 day after Photofrin injection at the same dose.34,35
Extensive measurements on low-dose-injected bladder cancer patients were made at the Department
of Urology, University of Leuven.33 The patients were injected with 0.35 or 0.50 mg/kg bodyweight, which
is a low enough dose to avoid sun sensitization, which, at therapeutic dose injection (≈2 mg/kg body-
weight), requires that patients be kept at reduced light level for about 4–6 weeks. Spectra for a papillary
bladder tumor are shown in Figure 6.9 for 337 and 405 nm excitation. It can be seen that there is a very
strong reduction in the blue-green fluorescence for tumor tissue, and also a prominent change in the
spectral shape when 337 nm is employed. This change allows malignant tumor detection without using
the HPD signal, which appears clearly only for 405 nm excitation, optimizing the conditions for HPD
detection while simultaneously reducing the autofluorescence intensity. Clinically, the most important
task is to be able to identify dysplasia and carcinoma in situ, and it was shown that such tissue has
sufficiently different spectral characteristics to be distinguished from normal tissue. Spectral curves for
mild and severe dysplasia are included in Figure 6.9. Data for tissues from 21 patients are shown in
Figure 6.10. Fluorescence monitoring on tumors of the prostatic gland has also been performed after
low-dose Photofrin injection and surgical resection. As can be seen in Figure 6.11, tumors are charac-
terized by an increase in the red fluorescence and a decrease in the blue-green fluorescence.36,37
The lung is, like the bladder, an important location where early tumor detection can have a major
influence on the course of disease. This is particularly true for patients with a positive sputum test but
negative biopsy sampling. We have studied the spectral characteristics of lung tissue during bronchoscopy
with regard to autofluorescence31 and after Photofrin injection.36,38 Spectra for a low-dose-injected patient
are shown in Figure 6.12.38
Tissue diagnostics based on fluorescence can sometimes be performed without any injection of a drug
by utilizing the properties of endogenous chromophores only. Early such work was performed by Chinese
researchers,39,40 particularly regarding the gastrointestinal tract, in which endogenous porphyrins fre-
quently localize in tumors. However, endogenous fluorescence can also be utilized to detect lung and
colon tumors, and also malignancies at many other locations.41–45
The unwanted side effect of skin sensitization can be avoided by using topical application in the case of
superficial tumors instead of systemic injection of a sensitizing drug. Topically applied δ-amino levulinic
acid (ALA) can be used for PDT of superfical skin tumors.46 ALA penetrates the damaged keratin in the
dermis but not the undamaged parts. ALA is a precursor in the biosynthetic pathway of the blood pigment
hemoglobin. ALA is a non-photoactive substance, but within the hem cycle it is transformed into proto-
porphyrin, which is a photodynamically very potent agent. This process is illustrated in Figure 6.13.47 The
steps from ALA to protoporphyrin appear comparatively fast, while the last step to hemoglobin is relatively
slow. Thus, an accumulation of protoporphyrin can be induced in the tumor cells. Subsequent exposure to
photoactivating light leads to a selective destruction of the lesions. In a first publication, a 90% response
rate for basal cell carcinoma following a single treatment was found.46 We have reported the same response
NORMAL 405 nm NORMAL 337 nm
FLUORESCENCE INT.
FLUORESCENCE INT.
TUMOR x 10
TUMOR TUMOR
450 500 550 600 650 700 750 350 400 450 500 550 600 650 700 750
WAVELENGTH (nm) WAVELENGTH (nm)
FLUORESCENCE INT. NORMAL
MILD DYSPLASIA
SEVERE DYSPLASIA
450 500 550 600 650 700 750

WAVELENGTH (nm)
FIGURE 6.9 Fluorescence spectra for a papillary bladder tumor and normal surrounding mucosa for a patient,
injected with a dose of 0.35 mg/kg bodyweight of Photofrin 48 hours prior to the investigation (top). Spectra are
shown for 337 and 405 nm excitation. Spectra for mild and severe dysplasia are also included.33
NORMAL 405 nm NORMAL 337 nm

FLUORESCENCE INT.
FLUORESCENCE INT.
TUMOR x 10
TUMOR TUMOR
450 500 550 600 650 700 750 350 400 450 500 550 600 650 700 750
WAVELENGTH (nm) WAVELENGTH (nm)
NORMAL
FLUORESCENCE INT.
MILD DYSPLASIA
SEVERE DYSPLASIA
450 500 550 600 650 700 750

WAVELENGTH (nm)
FIGURE 6.9 Fluorescence spectra for a papillary bladder tumor and normal surrounding mucosa for a patient,
injected with a dose of 0.35 mg/kg bodyweight of Photofrin 48 hours prior to the investigation (top). Spectra are
shown for 337 and 405 nm excitation. Spectra for mild and severe dysplasia are also included.33
rate for different kinds of superficial skin tumors, including basal cell and squamous cell carcinoma.47 The
conversion of ALA into protoporphyrin in tumors can be readily followed using laser-induced fluores-
cence.47,48 The superior tumor demarcation properties of ALA are illustrated in Figure 6.1447–49 for the case
NORMAL HUMAN LUNG SPECTRA 405 nm

DHE 1 mg/kg b.w. exc.
FLUORESCENCE INTENSITY
B fibre
TUMOR B3
400 450 500 550 600 650 700 750

WAVELENGTH (nm) A = 630 nm
B = 470 nm
FIGURE 6.12 Fluorescence spectra for a bronchial carcinoma and adjacent normal mucosa in a patient that had
received a dose of Photofrin at a concentration of 1 mg/kg bodyweight 48 hours before the fluorecence investiga-
tion.36,38
Glycine Haem hv
Succinyl Fe
CoA
ALA Feed- Singlet

back
Protoporphyrin O2
Porphobilinogen
Triplet
Protoporphyrinogen
Linear Tetrapyrrole
Coproporphyrinogen
Uroporphyrinogen
Tumor
Laser
630 nm
FIGURE 6.13 Schematic diagram of PDT using ALA. By exchanging the laser wavelength to about 400 nm and
recording the fluorescence from the tumor, its extent can be clearly determined.47
of a human basalioma. In the lower part of the figure, the corresponding fluorescence signals after PDT
(60 J/cm2) are shown. A strong photo-bleaching of the drug occurs, which can be used for dosimetry to
ascertain that the correct light dose has been delivered to all parts of the tumor.48 The bleaching of the drug
Pre PDT Pre PDT Pre PDT

80 Border 60 Tumor 70 Border
0 0 0
Pre PDT 500 600 700 500 600 700 500 600 700 Pre PDT
30 Normal 60 Normal
Tumor
17 mm
Normal skin Normal skin 0
0 500 600 700
500 600 700
20 Normal 30 Normal
0 0
500 600 700 500 600 700
Post PDT 10 Border 10 Tumor 10 Border Post PDT
0 0 0
500 600 700 500 600 700 500 600 700
Post PDT Post PDT Post PDT
FIGURE 6.14 Fluorescence data from scans through a basal cell carcinoma before (top) and after (bottom) PDT
(60 J/cm2). An ALA cream had been topically applied over the tumor 6 hours prior to the fluorescence investigation.47,49
in the upper strongly exposed tissue layer also allows a more efficient treatment of deeper lying layers by
continued light irradiation, because the upper layer cannot be overtreated.50
As already mentioned, there is an ongoing search for even better sensitizers, frequently concentrating
on the behavior in PDT procedures. Most sensitizers also have fluorescence properties that are useful for
tumor demarcation. A list of new sensitizers with wavelengths for PDT excitation and fluorescence
detection is given in Table 6.1.
TABLE 6.1 Photosensitizing Drugs Presently Under Different Stages of Clinical

Evaluation
Name Abbrev λ nm
Photofrin DHE 630

Bensoporphyrin BPD 690
δ-amino levulinic acid ALA 635
Mono aspartyl Chlorin e6 MACE 670
Tetra hydr. phenylchlorin THPC 650
Zinc phtalocyanine ZnPC 675
Tin ethyl etiopurpurin SnET2 660
Note: Common abbreviations and approximate wavelengths for PDT irradiation and
fluorescence detection are given.
6.3.4 Equipment for Imaging

It is of great diagnostic interest to extend fluorescence monitoring with incorporation of the contrast-
enhancement techniques illustrated in Figures 6.6 and 6.11 into imaging measurements. This has been
accomplished in our group, which has developed a multi-color fluorescence imaging system starting
with a one-dimensional proof-of-principle demonstration51 later extended into full two-dimensional
imaging.52–54 To simultaneously obtain spatial and spectral resolution, the fluorescent light is divided
using a multi-mirror arrangement as illustrated in Figure 6.15. By using a spherical mirror, which is
Trig CCD Camera

False
Signal
Color
Fiber
Image Coupler
Image
Video Intensifier
Signal HV
Supply
Split
Mirror
PC
Telescope
λ1
λ2 λ
λ4 3
Passband
Filters
Dichroic
Mirror
Quartz
Pulsed
Lenses
UV Source
Object
Plane
FIGURE 6.15 Diagram showing the basic construction of a multi-color fluorescence imaging system (From Ref. 53).
divided into four individually adjustable sectors, four identical images can be sent to an intensified
matrix detector, a CCD camera. By placing optical filters in front of the different mirror sectors,
fluorescence images in selected colors are obtained simultaneously. In the four different images,
computerized calculations of dimensionless function values can be performed for each spatial location
of the tissue investigated and a generalized image in an optimized contrast function can be produced.
This function would normally be the ratio between the background-free 630 nm peak intensity A and
the blue-green intensity B at about 470 nm. A properly weighted recording at 600 nm (kD) provides
the background intensity. The weighting factor k also takes differences in optical efficiency into account
and is adjusted to result in a zero intensity value for A = (A′-kD) when no tumor is present. A
photograph of a fully clinically adapted fluorescence imaging system based on the principles given
here is shown in Figure 6.16. This system can be used together with endoscopes of different types
allowing, e.g., lung and bladder investigations.
6.3.5 Multi-Color Fluorescence Imaging of Tumors

Fluorescence images at 630, 600 and 470 nm are shown in Figure 6.1754 for a human nodular basal cell
carcinoma. Also included is the resulting image using the contrast function (A′-kD)/B, showing the tumor
with a very clear demarcation toward surrounding normal tissue. A nitrogen-laser-pumped dye laser was
employed for fluorescence excitation. The data were obtained with a laboratory instrumentation requiring
substantial processing time to produce the final image. In an industrial prototype, a considerably higher
processing rate was achieved using a vector processor for image processing. Up to eight images per second
could be obtained allowing basic “live” recording of tumors. An example of a processed image from that
system is shown in Figure 6.18.56 This is a direct photograph of the monitor screen, showing a tumor,
in a rat that had been injected with Photofrin at a concentration of 15 mg/kg bodyweight. Registrations
of a malignant tumor in resected breast tissue, using the system shown in Figure 6.16, is given in
Figure 6.19.56 Here, several images are presented where the processed tumor image is superimposed on
a normal color video image using a video mixer. The patient had been given Photfrin at a dose of 0.35
mg/kg body weight 1 day before the operation. Further fluorescence imaging systems are described in
Refs. 57–60.
FIGURE 6.16 Photograph of a clincal multi-color fluorescence system adapted for bronchoscope applications (Cour-
tesy: Spectraphos AB, Lund).
FIGURE 6.17 Imaging fluorescence recordings of a nodular basal cell carcinoma on the head of a patient that had
been subject to topical ALA application 6 hours before the investigation. Individual images at 630, 600 and 470 nm
are shown together with a processed image using the data in the three individual images.55
FIGURE 6.18 Photograph of the computer monitor screen showing a processed image of an experimental rat
tumor of the same kind as was illustrated in Figure 6.5. The excitation source was a nitrogen laser, operating at
337 nm.55
6.4 Spectroscopic Diagnostics of Atherosclerotic Plaque
6.4.1 Basic Considerations

Cardiovascular disease is, besides cancer, the major cause of death in Western countries. Several risk
factors in the development of atherosclerosis, such as hypertension and smoking, have been identified.
When the process of atherosclerosis starts in the artery, the innermost part of the vessel wall, the intima,
with the endothelium cell lining is thickened and new constituents are deposited in the tissue. During
the initial stage, a fibrotic plaque develops, with increasing amount of collagen and elastin. Later in the
process of the disease, cholesterol crystals and lipid droplets are deposited. In the final stage, calcium is
incorporated and the former elastic vessel tube is transformed into a rigid, inflexible, partly obstructed
and brittle vessel. Sometimes, the surface of the plaque turns into a wound, which can easily be an area
for growth of dangerous blood clots. An atherosclerotically affected vessel is schematically shown in
Figure 6.20.61 The peripheral as well as coronary arteries can be affected by atherosclerotic disease. The
FIGURE 6.19 Imaging recordings of a malignant tumor in breast cancer. The instrument shown in Figure 6.16 was
utilized. The processed tumor image is superimposed on the normal color CCD output using a video mixer. Different
balances between the two types of images have been used.56
Vessel
lumen
Blood Stream
Intima
Media
Adventitia
Endothelium cell Cholesterol crystal

Smooth muscle cell Capillaries
Collagen
Lymphocyte
Macrophage Calcium
Lipid droplets Thrombosis
FIGURE 6.20 Schematic diagram of an atherosclerotically transformed vessel.61
change in chemical composition in the atherosclerotic vessel wall forms the basis for fluorescence diag-
nostics of this disease.
The surgical treatment of severe atherosclerosis includes aggressive procedures such as open-heart
surgery with coronary bypass. Several new modalities are now being investigated as possible replace-
ments for such extensive procedures. A new modality is laser-based transluminal angioplasty, in which
fiber-based catheters through a peripheral artery, e.g., the femoral artery, are used as an entrance for
intravasal treatment. A schematic diagram of such a procedure is shown in Figure 6.21.62 The field
is treated in detail in Chapter 116 of this volume. Different kinds of lasers have been used. Non-
thermal excimer laser ablation is of special interest for the treatment of coronary arteries, as thermal
Time-Integrated Diagnostics
EXCIMER LASER
Fluorescence Int.
Dichroic
Mirrors
Plaque
TISSUE SPECTRAL
DIAGNOSIS Normal
400 500 600

WAVELENGTH/nm
Time-Resolved Diagnostics
"Late"/"Early" Fluorescence
DIAGNOSTIC LASER Plaque
THRESHOLD
Normal
INTENSITY
0 5 10 15 (ns)
Aortic TIME
Plaque arch Plasma Emission Diagnostics
Normal
400 500 600
Left Emission Int.
Coronary
Heart
Ca and Ca+ Sodium

Lines D Line
Plaque
400 500 600
FIGURE 6.21 Schematic diagram showing transluminal laser ablation of atherosclerotic plaque with three varieties
of spectroscopic guidance: time-integrated fluorescence spectroscopy, time-resolved fluorescence monitoring and
laser plasma emission spectroscopy (From Ref. 62).
lasers induce artery spasm that might be avoided with UV ablation. Laser angioplasty is discussed in
Refs. 63–66.
Although promising, laser-based percutaneous angioplasty has some problems that must be overcome.
In addition to arterial spasm, vessel wall perforation and dissection are complications that must be
avoided. Another problem that follows all procedures performed in the vessel wall is restenosis, which
is caused by an overreaction from the treated tissue. This might be minimized by radical treatment.
Because the procedure is performed inside the vessel walls, a guiding system for the laser firing would
improve the possibility of monitoring the procedure and inhibiting the laser power when the ablation
has to be interrupted to avoid complications. A crude diagnostic signal is provided from the plasma
emission signal, as shown in Figure 6.21. A calcified plaque features strong lines of Ca and Ca+, which
disappear when the calcification has been ablated away.67,68
6.4.2 Plaque Discrimination Using Laser-Induced Fluorescence

It has been shown by several groups,69–73 including ours,28,68,74 that the fluorescence from the tissue itself,
the autofluorescence, can be used in demarcating atherosclerotic lesions from non-diseased vessel wall,
depending on the various chromophore content in the tissue types. The spectral shape of the tissue
fluorescence can be used in demarcating the lesions, as the shape reflects the different molecular contents.
Fluorescence provides a completely nonintrusive way of probing prior to firing a high-power pulse. When
excited with the light from a UV laser, the normal vessel wall emits strong fluorescence in the region
390–500 nm, while atherosclerotic plaque has a prominent peak at 390 nm, with a fall-off toward longer
wavelengths. This is illustrated by the spectra included in Figure 6.21. The signals are built up mainly
from contributions of elastin and collagen (see Figure 6.4a), with a relative increase in collagen in
atherosclerotic plaque being the origin of the spectral difference. In both spectra, a minimum in the
fluorescence signal can be seen at 420 nm, representing the strong absorption of hemoglobin in blood.
Weaker absorption peaks occur at 540 and 580 nm. The identification of these features becomes very
clear in a comparison with blood absorption spectra as shown in Figure 6.22.28 Clearly, the influence of
the blood reabsorption must be handled properly by a reliable fluorescence diagnostic system.
60
50
TRANSMISSION %
40
30
ARTERIAL BLOOD
20
10
0
400 500 600 700nm
50
40
30
20 VENOUS BLOOD
10
0
400 500 600 700nm
FIGURE 6.22 Absorption spectra for arterial and venous blood. Wavelength pairs with equal blood absorption are
indicated.28
F1 = F2 = F3 = F4 = F5 = F6 =
I(390) / I(480) I(415) / I(480) I(580) / I(600) I(390) / I(600) I(380) / I(437) I(390) / I(431)
1.5
6 3 30 3 3
1.0
4 2 20 2 2
0.5
2 1 10 1 1
IVIII II I 0 IVIII II I 0 IVIII II I 0 IVIII II I 0 IVIII II I 0 IVIII II I 0
FIGURE 6.23 Demarcation of five different types of vessel wall using fluorescence spectroscopy. Data for 6 different
demarcation criteria are shown. Criteria F5 and F6 are not affected by the presence of blood.28
In our investigations we have observed that the signature in vitro from a plaque region differs
depending on the stage of the atherosclerotic process. The early fibrotic lesions can be separated from
the plaque regions with higher fat content and also from calcified regions. We have been able to
spectroscopically separate four different classes of atherosclerotic lesions, depending on the severity
of the disease. This is illustrated in Figure 6.23.28 The intensity ratios at selected wavelengths have been
formed for a large number of samples. Group O is the normal non-diseased vessel wall tissue. Group
I includes the least damaged tissue, with only small amounts of fibrotic constituents. In group II, more
fibrotic constituents are present. Group III includes lesions with calcium content in the plaque, while,
in group IV, calcium occurs at the surface. In the latter group, the lesions with endothelial damage
with or without thrombotic material on the damaged surface are included. In the figure, all functions
except F3 show a substantial demarcation among the tissue types. Functions F5 and F6 are particularly
interesting, as they are immune to the presence of blood. This is because the wavelength pairs chosen
correspond to points of equal blood absorption, as indicated in Figure 6.22. Thus, in the ratio, the
influence of the absorption is largely eliminated.28,68,75
Similar techniques can be used for studying myocardial tissue for assessing the status of the heart
muscle. From a large number of biopsy samples, it was found that normal myocardial tissue can be
distinguished from scar or fatty tissue, as shown in Figure 6.24, where two fluorescence intensity ratios
have been used for the demarcation.76
5 R1 390/465
4,5
4
3,5
3
2,5
2
1,5 NORMAL
1 FAT
,5 SCAR
0
,5 1 1,5 2 2,5 3 3,5 4 4,5
R3 465/525
FIGURE 6.24 Diagram showing differentiation of different kinds of myocardial tissue using fluorescence (From
Ref. 76).
FIGURE 6.25 Photograph of a fluorosensor for plaque monitoring employing photomultipliers observing fluores-
cent light, that has been selected for wavelength by interference filters (Courtesy: Spectraphos AB).
Some in vivo investigations were performed in connection with coronary bypass procedures using a
clinically adapted fluorosensor system, as shown in Figure 6.25. The system displays the ratio of the signal
intensities for the blood-independent wavelength pair 380 and 440 nm. The fiber probe of the system
was placed where the coronary arteries were opened to be attached to the new vessels. Investigations were
also performed inside the coronary arteries in the peripheral direction, with the probe inserted into the
I(380)
I(380) RATIO =
I(440)
I(440)
RATIO
NORMAL PLAQUE NORMAL PLAQUE NORMAL
FIGURE 6.26 Recording from the instrument shown in Figure 6.25 for normal tissue and plaque in human aorta.77
(a) (b)
Intensity (relative units)

Normal vessel Normal tissue
5 10 15 10 20 30
Time (ns) Time (ns)
Plaque Malignant tumor
5 10 15 10 20 30
Time (ns) Time (ns)
FIGURE 6.27 Time-resolved fluorescence decay curves for a) atherosclerotic plaque and normal vessel wall, and b)
an experimental tumor in an Photofrin-injected animal, and normal surrounding tissue.75,78
lumen toward known occlusions. Fluorescence was also measured from normal aortic wall close to the
heart. A recording using this system is shown in Figure 6.26.77
6.4.3 Time-Resolved Monitoring

In all the fluorescence measurements discussed so far, the total fluorescence intensity following pulsed
laser excitation has been used. However, the temporal decay characteristics of the fluorescent light can
also be utilized for tissue characterization, e.g., for distinguishing atherosclerotically diseased vessel wall
from normal vessel wall.12,28,75,78 The fluorescence from the diseased region has a longer decay time than
the normal vessel wall tissue, as shown in Figure 6.27. This is basically because collagen, more abundant
in plaque, has a longer lifetime than elastin.13 These recordings were taken with an advanced set-up
incorporating a mode-locked picosecond laser source in conjunction with time correlating photon-
counting electronics. By a detailed analysis, it is possible to unfold the decay into three components with
different decay times.78 However, since the lifetimes involved are of the order of ns, for a clinically practical
device it is also possible to use a normal nitrogen laser with a pulse duration of about 3 ns and to use
gated integrators to capture “late” fluorescence (5–15 ns) and divide it by “early” fluorescence (0–5 ns).
A recording obtained in this way when alternating the fiber tip between plaque and normal wall is
included in Figure 6.21. Because a single detection wavelength is used and because blood is non-fluo-
rescing, the data obtained are immune to the presence of blood, if the blood layer is not so thick that
the whole signal is blocked out. A system based on the spectroscopic characterization of diseased tissue
for guiding purposes in percutaneous laser angioplasty could, of course, include time-integrated as well
as time-resolved aspects to improve the classification ability. In fact, an instrumentation of the type shown
in Figure 6.25 allows both these aspects to be utilized.
Decay curves detected at 630 nm for an experimental tumor and surrounding normal issue are also
included in Figure 6.27. The animal had earlier been injected with Photofrin. It can be seen that the
tumor curve has a fast and a slow decay component, while normal tissue has only a fast decay. The fast
component corresponds to the tissue autofluorescence, whereas the more long-lived component is due
to the HPD drug. As was noted in Ref. 28, gating on late fluorescence yields only suppression of the
autofluorescence and contrast enhancement using a single detection wavelength.
6.4.4 Raman Spectroscopy for Tissue Diagnostics

Raman spectroscopy has long been a powerful tool used by chemists in the analysis of complex organic
molecules. The techniques of Raman spectroscopy are now being adopted for tissue spectroscopy.79–81 As
mentioned above, the main difficulty with this technique is the weakness of the signals and the competing
process of fluorescence, which normally tends to completely swamp the Raman signals. An efficient way
to reduce the influence of fluorescence is to use a laser with a near-IR wavelength, which does not excite
fluorescence very efficiently. A CW Nd:YAG laser (λ = 1064 nm) in combination with a Fourier transform
spectrometer, provides an efficient way of recording Raman spectra.82 A clinically more practical way is
to use a near-IR diode laser in combination with a cooled near-IR CCD array.80 A different way to suppress
fluorescence is to use a picosecond laser source and limit the detection to the duration of these short
pulses. Then fluorescence, which typically has a lifetime of a few ns, is reduced while all the Raman
photons are recorded, because the latter process is basically instantaneous. This procedure can, however,
hardly replace the use of a near-IR laser.
Raman spectra of plaque and its molecular constituents are particularly impressive. Such recordings
are shown in Figure 6.28.81 Here, near-IR Fourier transform spectra of fibrous plaque, atheromatous
plaque and cholesterol powder (bottom) are shown, featuring strong Raman peaks corresponding to the
vibrational shifts. The 1667 cm-1 vibrational mode from C = C (stretch), and the 1440 cm-1 mode of C-
H (bend) are particularly prominent. Raman spectra are also being investigated for the diagnosis of
malignant tumors; the results seem promising. Through the increased specificity of Raman spectra over
fluorescence spectra, an interesting potential for powerful tissue diagnosis exists. Because of the weak
signals and the need for a respectable spectral resolution, the possibilities for real-time Raman tissue
imaging are limited.
6.4.5 Tissue Reflectance Monitoring

We have already discussed visible light tissue reflectance in Section 6.2.1 and noted that this is the normal
means by which color on the object is determined. However, by going toward the IR region, much more
specific information can be obtained, as molecular overtone vibrational transitions occur here, making
an imprint in the reflectance spectrum. Thus, in a certain sense, information similar to Raman spectros-
copy can be obtained in IR reflectance spectroscopy. This has been used for tissue spectroscopy.83
6.5 Light Scattering and Tissue Transillumination

The basic principles of elastic light scattering in tissue were discussed in Section 6.2.5. Light scattering
in tissue complicates normal light propagation, as straight beam lines cannot be obtained. Thus, mea-
surements of tissue oxygnation using differential light absorption at the near-IR absorption peaks of
hemoglobin cannot assume a well-defined path length through the tissue probed, but a considerably
1448
1662
(a)
1265
1340
1005
1440
Intensity (arb. units)
1667 (b)
1300
701
1130
959
882
1439
1671
(c)
1328
700
1130
957
878
1800 1600 1400 1200 1000 800 600 400

-1
Frequency (cm )
FIGURE 6.28 Raman spectrum of (a) fibrous plaque, (b) atheromatous plaque and (c) cholesterol monohydrate
powder, excited with a 1064 nm CW Nd:YAG laser.81
longer effective path length is obtained. Also, in transillumination, no sharp images can normally be
obtained because of the strong scattering. Using time-resolved spectroscopy, it is possible to follow the
photon paths through the tissue. Two basic geometries are illustrated in Figure 6.29.84 In transillumina-
tion, there is a shortest path corresponding to the very unlikely event of non-scattered radiation, giving
rise to a distinct signal (ballistic photons). Most photons arriving at the detector come much later, and
only after several scattering events. If the transmitter and the detector are at the same side of the sample,
all photons received must have been scattered more or less. If the position of transmitter and receiver
coalesce, a histogram of arrival times for photons that have sampled a certain volume below the mea-
surement point is obtained. This mode of operation is common in Doppler measurements of superficial
blood flow, which we will discuss first.
6.5.1 Doppler-Flow Monitoring

A measure of the blood perfusion in tissue can be obtained by analyzing the signal due to slightly
Doppler-shifted photons that encountered scattering against moving red blood cells. In such mea-
surements, normally a CW helium-neon laser operating at 633 nm, or a dark red near–IR diode
laser is used. Obviously, an objective full quantification is difficult to obtain, because a complex
averaging is performed over photons scattered at different depths. Still, using experience, valuable
information can be obtained, in particular for assessing reperfusion in connection with transplant
surgery. The techniques and clinical application of laser Doppler blood flowmetry can be found in
Refs. 20 and 21.
a)
A A B
Time Time
b) X
Absorbing Tumor Input Pulse
Time-Integrated light
Time
Intensity
Time-Gated light
FIGURE 6.29 (a) Illustration of time-resolved signals received in elastic photon scattering measurements on tissue
using two different geometries. (b) Enhanced viewing through a scattering medium using detection of the first
arriving photons in transillumination (From Ref. 84).
By performing sequential measurements with a system where the laser beam is scanned in a matrix
pattern over the tissue, it is possible to generate images of the superficial blood flow.86 A schematic
diagram of such a system is shown in Figure 6.30.86 An example of the application of such a system is
shown in Figure 6.31,87 where the blood flow in a tumor region is shown in connection with PDT. A
basal cell carcinoma was treated after topical application of ALA. The perfusion image is shown on four
occasions. First, an image before the treatment was taken. Directly after PDT (60 J/cm2), given at such
a low laser light fluence rate that tissue heating is avoided, an increased blood flow is seen in the tumor
and surrounding area, indicating an immediate inflammatory tissue response. One week after the treat-
ment, the central tumor area is covered by a crust surrounded with a high perfusion area. Finally, 8.5
months after the treatment, a renewed imaging of the area shows a reduction of the blood flow in the
tumor to the same level as the surrounding normal tissue.
FIGURE 6.30 Arrangement for scanning laser Doppler flowmetry for tissue.86
FIGURE 6.31 Imaging measurements of superficial blood perfusion in connection with ALA PDT. Recordings are
shown for the following situations: a) before PDT, with the tumor still covered by a crust, b) after removal of the
crust, c) immediately after PDT, and d) 2 weeks after treatment.87
6.5.2 Time-Resolved Scattering Spectroscopy

As mentioned above, time-resolved transillumination allows enhanced viewing through tissue. X-ray
diagnostic techniques are the conventional means for such imaging that has been brought to a very high
level of sophistication. However, ionizing radiation is accompanied with a certain risk of mutagenicity.88
It would be very desirable to use optical radiation for transillumination of tissue to avoid this risk and
also to allow spectroscopic recordings of molecular-specific absorption. Red light penetrates tissue quite
well, as can be evidenced when shining light from a flashlight through the hand. The reason is the quickly
decreasing absorption of hemoglobin beyond 600 nm, as is illustrated by the corresponding curve in
Figure 6.1. The low intensity of the transmitted light through thick tissue is not a problem, as very
sensitive light detectors exist, but rather, the strong multiple scattering from the cell structures, washing
FIGURE 6.32 Transillumination scan through a human hand with data for the total time-integrated light and the
early light.94
out all the spatial information. Scattering prevents the observation of the bones in the hand as shadows.
The scattering has limited the application of optical transillumination (diaphanography) for mammo-
graphic investigations.89–91 Only superficial tumors close to the skin in the breast parenchyma can be
made visible. Recently, much research has been initiated to overcome these problems,85,92 e.g., by using
time-resolved photon-counting techniques employing picosecond red laser pulses.93–96 Because of the
multiple scattering, most of the photons penetrating the tissue have spent a long time in the tissue, while
only very few photons arrive to the detector at the nominal propagation time without disruptions.
However, by selecting a very small detection time window and recording the very first photons, it is
possible to look at just the “fastest” photons coming out of the tissue. If an obstacle, such as a bone or
a tumor blocks the passage, a shadow will be created for the selected subgroup of very early photons.
For thicker samples, there are no unscattered photons. Still, the first photons have been minimally
scattered, producing a clearer image, as schematically shown in Figure 6.29b. An example of a scan
through a human hand is shown in Figure 6.32.94 A cavity-dumped dye laser, synchronously pumped by
an argon-ion laser, was used in these measurements. Time-gated single-photon counting was used to
record “early” photons in a time window 80 ps wide. Clear shadows of the bones can be seen in the time-
gated scan, while the contrast disappears in the time-integrated recording.
6.5.3 Transillumination Imaging

By scanning the tissue under investigation, a transillumination image can be created. Recently, we have
shown that such measurement can also be performed using a pulsed diode-laser source, making the
FIGURE 6.33 Two-dimensional transillumination scans through a resected breast sample containing a ductal car-
cinoma. The tumor clearly emerges in the early light, while it remains hidden in time-integrated monitoring.96
X - Y Translator
Glass
Slide
Diode Laser
Driver Laser
Sample Optical
Fibre
CFD Reference
Fibre
TAC CFD Amp Detector
PC
MCA
FIGURE 6.34 Set up for two-dimensional time-gated transillumination imaging through tissue, using a pulsed diode
laser and time-correlated photon counting electronics.96
technique very realistic.96 Images of a ductal breast carcinoma in a newly resected breast are given in
Figure 6.33. A diode laser, operating at 815 nm and producing 30 ps-long pulses at a repetition rate of
10 MHz was used in these recordings, performed with the set-up shown inFigure 6.34. The transmitting
and receiving fibers are scanned together under computer control over the tissue, which is compressed
between glass plates. In the time-integrated light, the tumor cannot be seen. It was also difficult to detect
using conventional x-ray mammography. In the time-gated image, the tumor can be clearly seen. To
eliminate possible artifacts, it is advantageous to divide the early light by the total light, as also shown
in the figure.
Many groups are now working in this new field of research, developing a variety of techniques for
suppressing excessive scattering using streak-camera,97 Raman-amplifier98 and frequency-doubling gat-
ing.99 Fast modulation of the source followed by detection of phase shifts and loss of modulation depth
can give similar information.100–102 The coherence properties of non-scattered light are used in
heterodyne103 and light-in-flight holography.104,105 Imaging in scattering media can also be performed
with spatial domain reflectometry.106,107
6.5.4 Interpretation of Time-Resolved Tissue Recordings

This section will focus on the intepretation of time-resolved recordings of tissue to understand, e.g.,
how tumors can be detected in the early light. This is best performed by examining the results of a model
experiment95 performed with picosecond pulses traversing a 30 mm-thick cuvette containing a mixture
of Intralipid (Kabi Vitrum) and ink in water. Intralipid is a strongly scattering, non-absorbing liquid,
while ink has a strong absorption but negligible scattering. By mixing these two substances, the values
of the scattering and absorption coefficients, µs and µa, can be varied freely. In the upper part of
Figure 6.35, time-resolved recordings can be seen, where no ink but increasing amounts of Intralipid
have been added. It can be seen that, at early times, the time dispersion curves are strongly influenced,
while the “decay constant” at late times is little affected. If, on the other hand, increasing amounts of ink
are added for a constant amount of Intralipid, it can be seen in the lower part of Figure 6.35 that the
early light is basically unaffected, while the “decay constant” for the late light is quickly shortened for
increasing absorption. In the figure, an early fixed detection window is indicated. Using these curves, the
increasing signal for a tumor in Figure 6.33 (brighter areas) can be interpreted as a reduction in the
scattering in a tumor rather than a change in the absorption. This means that the early light is not useful
for absorption spectroscopy changing the wavelength. On the other hand, wavelength-dependent scat-
tering changes might prove useful for tissue identification. The decay constant for the late light is related
in a simple way to the absorption coefficient.108 However, for this “late” light, little image information is
retained. By using the whole temporal dispersion curve, maximal tissue information can be extracted
using wavelength-dependent properties of different chromophores and tissue structures.
Focusing on the properties of the late light, spectroscopic analysis has been performed15 for assessing
the concentration of photosensitizers in tumors for PDT light dosimetry. Then, light with a wavelength
on the sensitizer absorption peak, as well as light off that peak, is analyzed. The first time-resolved
measurements on human tissue concerned brain oxygenation assessment, e.g., in premature infants, by
using differences in oxy- and deoxyhemoglobin absorption108–110 (see Figures 6.1 and 6.23).
At the end of this section, we should note that tissue information can be obtained also when time-
integrating instrumentation is used, e.g., when a CW light source is employed. As we have noted, the
pathlength is not well defined, due to multiple scattering in the tissue. Still, equipment used in a
standardized way can yield reliable data. An example of this is the blood oxygenation measurements
performed with a pulse oximeter, where light is sent through a patient’s fingertip, probing the differential
absorption of oxy- and deoxyhemoglobin.111 Normally, light at three different wavelengths is used,
generated by light-emitting diodes or diode lasers. 805 nm provides a reference point, where oxy- and
deoxyhemoglobin absorb equal amounts (the isobestic point). At 660 and 940 nm, the two types of
hemoglobin have very different absorptions. The oxygenation value (oxygen tension) obtained with a
pulse oximeter has been shown to be influenced by the hematocrit value (density of red blood cells) and
by complex formation in the blood of smokers. Even if the absolute value is subject to various influences,
the instrument has an important application in monitoring of patient status from the time before
anesthesia through an operation, or during bronchoscopy. A further example is a technique for cancer
diagnostics, where the elastically scattered light from a broadband source is analyzed at some distance
from the point of light injection into the tissue.112 Because the scattering properties vary for different
types of tissue, the signal recorded can be compared with catalogue spectra obtained for well characterized
tissues. Thus, a tissue diagnosis can be obtained in this empiric way.
6.6 Outlook
As illustrated in this chapter, many new possibilities for tissue diagnostics using lasers are emerging,
addressing important fields such as cancer detection, cardiovascular monitoring and tissue transillumi-
µa 0 mm-1
A
µs(1-g)=0.30 mm-1
µs(1-g)=0.45 mm-1
Intensity (Arb. Units)

µs(1-g)=0.60 mm-1
µs(1-g)=0.75 mm-1
µs(1-g)=0.90 mm-1
0 0.5 1 1.5 2 2.5

120 psec time-gate Time (nsec)
B µs(1-g)=0.38 mm-1
µa 0 mm-1
µa=0.0045 mm-1
Intensity (Arb. Units)
µa=0.009 mm-1
µa=0.014 mm-1
µa=0.018 mm-1
0 0.5 1 1.5 2 2.5

120 psec time-gate Time (nsec)
FIGURE 6.35 Experimental time-dispersion curves for light propagation through a 30 mm thick cuvette filled with
a scattering and absorbing liquid. From the curves it is evident that the amount of early light is mainly influenced
by the scattering and not by the absorption.95
nation imaging without using ionizing radiation. The rapid development of more and more practical
and cost-effective laser sources, detectors and computers facilitates the clinical implementation of the
new techniques. The noninvasive nature of the techniques and the real-time data acquisition are attractive
features of the optical methods. Optical spectroscopy, in principle, allows molecular analysis, providing
information not normally accessible with other modalities. For the future, an increased use of combined
diagnostic and therapeutic equipment can be anticipated. Ideally, each single cell should be optically
diagnosed, followed by an immediate decision on whether that cell should be eliminated through the
immediate use of laser ablation, heating or photochemistry.
Acknowledgments
The author gratefully acknowledges a most stimulating collaboration with a large number of colleagues
and graduate students within the Lund University Medical Laser Center, as well as with many foreign
groups. This work was supported by the Swedish Research Council for Engineering Sciences, The Swedish
Board for Technical and Industrial Development, the Swedish Cancer Foundation and the Swedish
Medical Research Council.
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7
Low-Power Laser Effects
7.1 Introduction ....................................................................... 171

7.2 Primary and Secondary Mechanisms of the Action
of Monochromatic Visible and Near Infrared
Radiation on Cells .............................................................. 172
Photoacceptors for Low-power laser Effects • Primary
Mechanisms of Light Action • Secondary Reactions of
Light Action
7.3 Explanation of Controversies and Limitations
of Low–Power Laser Effects on Cellular Level ................. 187
Magnitude of Low-Power Laser Effects • Diversity of Low-
Power Laser Effects • Biological Limitations of Low-Power
Laser Effects • Dependence on the Physiological Status of the
Host Organism
7.4 Clinical Applications of Low-Power Laser Effects ........... 196
Focus of Application • Is Low-Power Laser Therapy a Mature
Modality? • Possible Optimal Light Parameters for Low-Power
Tiina Karu Laser Therapy
Laser Technology Research Center 7.5 Summary............................................................................. 200
of Russian Academy of Science References ...................................................................................... 201
7.1 Introduction
The most frequently used mechanism of photon energy conversion in laser medicine is heating. Average
heating of irradiated samples occurs with all methods of tissue destruction (cutting, vaporization, coag-
ulation, ablation). Many of these surgical laser techniques are reviewed elsewhere in this book.
At low light intensities, the photochemical conversion of the energy absorbed by a photoacceptor
prevails. This type of reaction is well known for specialized photoacceptors such as rhodopsin or
chlorophyll. In medicine, light absorption by non-specialized photoacceptor molecules (i.e., molecules
that can absorb light at certain wavelengths, but are not integral to specialized light reception organs)
is used rather extensively (Figure 7.1). The absorbing molecule can transfer the energy to another
molecule, and this activated molecule can then cause chemical reactions in the surrounding tissue.
This type of reaction is successfully used in photodynamic therapy (PDT) of tumors (also discussed
elsewhere in this book). Alternatively, the absorbing molecule in a light-activated form can take part
in chemical reactions, as occurs in treatment of skin diseases with psoralens and UVA radiation (PUVA).
Importantly, in both PDT and PUVA therapy, the photoabsorbing molecules are artificially introduced
into a tissue before irradiation.
Irradiation of cells at certain wavelengths can also activate some of the native components. In this way,
specific biochemical reactions, as well as whole cellular metabolism, can be altered. This type of reaction
is believed to form the basis for low-power laser effects (Karu, 1989b; Smith, 1991). One should note
that light therapy methods, based on photochemical conversion of photoabsorbing molecules
0-8493-1146-2/02/$0.00+$1.50
LOW-POWER (LASER) LIGHT IN MEDICINE
[ exogenous (artificially [ endogenous (natural)

introduced) photoacceptors] photoacceptors]
PHOTODYNAMIC THERAPY (PDT) PHOTOTHERAPY WITH UV
PHOTOCHEMOTHERAPY (PUVA) LOW-POWER LASER THERAPY

(LASER BIOSTIMULATION)
FIGURE 7.1 Methods of light therapy based on endogenous and exogenous photoacceptors in cells.
(Figure 7.1), are not laser-specific methods. Conventional sources generating the appropriate wavelength
can also be used (as is done in PUVA and UV therapy). Laser sources are just handy tools providing
many practical advantages (e.g., efficient fiber optic coupling to irradiate interior body parts, high
monochromaticity and easy wavelength tunability, simplicity in use and electrical safety in the case of
semiconductor lasers). However, for therapeutic effect in deeper tissue levels, coherent light could provide
some additional benefits (Karu, 2002).
Low-power laser effects are the topic of the present chapter. During the past few years, numerous
reviews on this topic have been written describing various aspects of the problem: history (Karu,
1987,1989a), controversies (Karu, 1989a; Smith, 1991; Belkin et al., 1988; Harris, 1988; Berns and Nelson,
1988; Basford, 1989), quantitative laws of visible light action on cells (Karu, 1987, 1989b, 1991a), pho-
tobiological fundamentals (Karu, 1989a, 1998), molecular mechanism (Karu 1988). Specific data about
the effects of irradiation on various cells has also been reviewed, including: cell cultures in vitro
(Karu,1990,1991b), Escherichia coli (Tiphlova and Karu, 1991a), microorganisms (Karu, 1996a), and
human lymphocytes (Karu, 1992b, 1996b). In addition, clinical applications of low-power laser therapy
have been surveyed (Ohshiro and Calderhead, 1988, 1991; Calderhead et al., 1991; Baxter, 1994; Tunér
and Hode, 1999). As a rule, the material from those previous reviews is not repeated here.
The present review is designed in the following way. First, primary and secondary mechanisms of
action of visible and near IR radiation on cells are described (Section 7.2). Second, controversies and
limitations are considered (Section 7.3). Third, a short glance into clinical applications of low-power
laser therapy is presented (Section 7.4).
7.2 Primary and Secondary Mechanisms of the Action

of Monochromatic Visible and Near Infrared Radiation
on Cells
A photobiological reaction involves the absorption of a specific wavelength of light by the functioning
photoreceptor (photoacceptor) molecule. To distinguish specialized photoreceptor molecules such as
rhodopsin, phytochrome, bacteriorhodopsin and chlorophylls from nonspecialized chromophores, the
photoacceptors take part in a metabolic reaction in a cell that is not connected with a light response.
After absorbing the light of the wavelength used for irradiation, this molecule assumes an electronically
excited state from which primary molecular processes can lead to a measurable biological effect in certain
circumstances. To work as a photoacceptor taking part in photobioregulation, this molecule must be part
of a key structure that can regulate a metabolic pathway. Redox chains are example of this type of key
structures, which suit to these requirements.
7.2.1 Photoacceptors for Low-Power Laser Effects

Several pieces of evidence show that mitochondria are sensitive to irradiation with monochromatic
visible and near infrared (IR) light. The illumination of isolated rat liver mitochondria increased
adenosine triphosphate (ATP) synthesis and the consumption of O2 (Kato et al., 1981; Passarella et
al., 1984; Gordon and Surrey, 1960). Irradiation with light at wavelengths of 415 nm (Kato et al.,
Low-Power Laser Effects 173
1981), 602 nm (Vekshin and Mironov, 1982), 632.8 nm (Passarella et al., 1984), 650 nm and 725
(Gordon and Surrey, 1960) enhanced ATP synthesis. Light at wavelengths of 477 and 554 nm (Kato
et al., 1981) did not influence the rate of this process. Oxygen consumption was activated by illumi-
nating with light at 365 and 436 nm, but not at 313, 546 and 577 nm (Vekshin and Mironov, 1982).
Irradiation with light at 633 nm increased the mitochondrial membrane potential (∆ψ) and proton
gradient (∆pH), caused changes in mitochondrial optical properties, modified some NADH-linked
dehydrogenase reactions (NADH is a reduced form of nicotinamide adenine dinucleotide) (Passarella
et al., 1983) and increased the rate of ADP/ATP exchange (ADP is adenosine diphosphate) (Passarella
et al., 1988a) as well as RNA and protein synthesis in the mitochondria (Hilf et al., 1986). In the case
of state 4 respiration, the 351 nm and 458 nm laser irradiations accelerated the oxygen consumption
of rat liver mitochondria; such acceleration was not observed with 514.5 nm irradiation. On the
contrary, in the case of state 3 respiration, the 514.5 nm laser irradiation activated the oxygen con-
sumption of mitochondria. Activation did not occur with 458 nm irradiation, and 351 nm irradiation
reduced the oxygen consumption in state 3 (Morimoto et al., 1994). The 660 nm irradiation increased
state 3 oxygen consumption at both coupling II and III sites, as well as increasing the respiratory
control ratio (Yu et al., 1996).
It is also believed that mitochondria are the primary targets when the whole cells are irradiated with
light at 630 nm (Hilf et al., 1986), 632.8 nm (Karu et al., 1995a; Bakeeva et al., 1993; Manteifel et al.,
1997) or 820 nm (Herbert et al., 1989). Irradiation with light at 812 nm (Loevschall and Arenholdt-
Bindslev, 1994a) or 632.8 nm (Anders et al., 1995) altered the rhodamine 123 uptake by fibroblasts. These
results were interpreted by the authors as inducting the perturbation of mitochondrial energy production
(Loevschall and Arenholdt-Bindslev, 1994a) and membrane potential (Anders et al., 1995).
The question is, which molecule in a mitochondrion is responsible for the effects mentioned above?
When considering the cellular effects, this question can be answered with the aid of action spectra. We
know that, within certain limits, an action spectrum follows the absorption spectrum of the photoacceptor
molecule (Hartman, 1983). On the other hand, the action spectrum is insufficient to distinguish between
potential photoacceptor pigments with very similar absorption. Moreover, the absorption spectrum for
the photoacceptor pigment may depend strongly on its environment, but this environment remains
unknown as long as the photoacceptor pigment itself is unknown. Because of these inadequacies of the
photoacceptor pigment, some other criteria for identification are also used.
Action spectra for the DNA and RNA synthesis rate in HeLa cells of the exponential and plateau phase
of growth, as well as those for the adhesive properties of HeLa cellular membranes, were published by
Karu et al. (1984a,b, 1996a). Figure 7.2 shows a generalized action spectrum for HeLa cells (a sum of the
four spectra from the paper of Karu et al. (1984a,b). Recall that in the wavelength range 310–500 nm, a
maximum stimulating effect was obtained with a radiation dose one order of magnitude less than in the
longer-wave spectral range (Karu et al., 1984a, b).
Figure 7.2 shows that the action spectrum in the range 580–860 nm consists of two series of doublet
bands in the range 620–680 nm and 760–895 nm with well-pronounced maxima at 620, 680, 760
and 825 nm. In the violet-blue region there is one maximum at 400 nm with the edge of the envelope
near 450 nm.
It is known that the action spectrum is roughly the same shape as the absorption spectrum of the
photoacceptor (Hartman et al., 1983). Therefore, the bands in the action spectra were identified by
analogy with the metal-ligand systems absorption spectra characteristic of this spectral range (Wilkinson
et al., 1987; Siegel, 1971–1981; Hughes, 1987). The regions 400–450 nm and 620–680 nm are characterized
by the bands pertaining to complexes with charge transfer in a metal-ligand system, and within 760–830
nm, these are d-d transitions in metals. The region 400–420 nm is typical of π-π* transitions in a
porphyrin ring (Gouterman, 1978).
Comparative analysis of spectral data for transition metals and their complexes on one hand, and
biomolecules participating in the regulation of cellular metabolism on the other, allows us to suggest
that multinuclear enzymes containing Cu(II) may be participating (Hughes, 1987; Lichtenstein, 1979).
Analysis of the electron excitation transitions of participating molecules containing Cu(II) (Hathaway,
FIGURE 7.2 The generalized action spectrum of proliferation increase of HeLa cells for λ = 330–860 nm (Karu et
al., 1984a, b). Curve 1-dose 10 J/m2, curve 2-dose 100 J/m2. A possible belonging of peaks to absorbing chromophores
is marked as suggested by Karu and Afanasyeva, 1995.
1987; Lontie, 1984; Spiro, 1981; Karlin and Zubieta, 1983) shows that metal-ligand transitions in the
range at 400–450 nm correspond to the Nimidasole → Cu transition, at 620 nm, to Scysteine → Cu transition,
and at 680 nm to Smethionine → Cu transition.
Comparing the lines of possible d-d transitions and charge-transfer complexes of Cu (Hughes, 1987;
Lontie, 1984; Spiro, 1981; Karlin and Zubieta, 1983; Brunori and Chance, 1987) with our action spectrum
(Figure 7.2) allows us to assume that the photoacceptor molecule has different types of centers containing
Cu(II) in the ranges 420–450 nm and 760–830 nm. In the range 420–450 nm, this may be a combination
of centers of types I and II (for the characteristics of centers of types I, II and III see Lichtenstein, 1979)
though a center of type I may be present. At 330 nm, a center of type III may be present, and in the
range 760–820 nm centers of types I and III coexist. Within 620–680 nm, there is a center of type I and
a combination of centers of different types is unlikely.
The above analysis allows us to conclude that all bands in the action spectrum in Figure 7.2 may be
related to the cytochrome c oxidase. The fact that the photoacceptor is a component of the respiratory
chain was considered earlier (Karu, 1989a). Cytochrome c oxidase (or cyt a/a3), is the terminal enzyme
of the respiratory chain in eukaryotic cells (Figure 7.3a), which mediates the transfer of electrons from
cyt c to molecular oxygen. Ferrocytochrome c is oxidized, dioxygen is reduced, and protons are pumped
vectorially from the mitochondrial matrix to the cytosol. Free energy resulting from this redox chemistry
is converted into an electrochemical potential across the inner membrane of the mitochondrion, which
ultimately drives the production of ATP. Accordingly, cytochrome c oxidase plays a central role in the
bioenergetics of the cell.
Cytochrome c oxidase of mammalian cells is a large multicomponent membrane protein of consid-
erable structural complexity. Two heme moieties (heme a and heme a3), two redox active copper sites
(CuA and CuB), one zinc and one magnesium, are located in 13 subunits (molecular size of 200
kilodaltons). Recently, the high resolution three-dimensional x-ray structure of cytochrome c oxidase
of bovine heart (Tsukahara et al., 1995, 1996) and Paracoccus denitrificans (Iwata et al., 1995) were
reported. These studies indicated that CuA is a dinuclear copper center with an unexpected structure
similar to a [2Fe-2S] type iron-sulfur center in which the Fe ions and inorganic sulfur atoms are
replaced with Cu ions and cysteine sulfur atoms respectively. The O2 binding site contains heme a3
FIGURE 7.3 (a) Mitochondrial respiratory chain of eukaryotic cells. (b) A scheme of the catalytic cycle of cyto-
chrome c oxidase (after Gennis and Ferguson-Miller, 1995).
iron and CuB; there is no detectable bridging ligand between iron and copper atoms. Heme a is
coordinated with two imidazoles of histidine residues. The fifth ligand of heme a3 is an imidazole,
whereas CuB is coordinated by three imidazoles of histidine. CuA (Cu-Cu) center is coordinated by
residues of two cysteins, two histidines, one methionine and one peptide carbonyl of a glutamate
(Tsukahara et al., 1995).
In the catalytic cycle of cytochrome c, oxidase electrons are transferred sequentially from water-soluble
cytochrome c to CuA, then to heme a and to the binuclear center a3-CuB, where oxygen is reduced to
water (Figure 7.3b). Oxygen binds to heme a3 and is reduced to water through a series of short-lived
elusive intermediates. Singular value decomposition analysis indicated the presence of at least seven
intermediates (Sucheta et al., 1997). The best characterized species until now are ferrous-oxycomplex
and peroxy species (Babcock and Wikström, 1992; Verkhovsky et al., 1996; Sucheta et al., 1997).
Generally speaking, the cytochrome c oxidase can be fully oxidized (four redox active metal centers:
CuA, CuB, irons in hemes a and a3, are in their common higher oxidation state; 3+ for iron and 2+ for
copper), or fully reduced (four metal centers are in their common lower oxidation state; 2+ for iron and
1+ for copper). Partially reduced enzymes, usually called mixed-valence one, have some metal centers in
their higher oxidation state and the remainder in their lower oxidation state. There are also a number
of forms of oxidized enzyme: fast enzyme (reacts relatively rapidly with cyanide), slow enzyme (reacts
at about 1% of the rate of the fast enzyme, also called resting enzyme), pulsed enzyme (obtained by
reducing slow enzyme and oxidizing it with oxygen under conditions in which the production of H2O2
is avoided), oxygenated enzyme (subjected to a cycle of reduction and reoxidation under conditions in
which H2O2 is produced) (Capaldi, 1990; Palmer, 1993). These details are given to illustrate how com-
plicated and controversial the overall picture of cytochrome c oxidase functioning still is.
Coming back to the comparative analysis of the action spectrum in Figure 7.2 and available spectro-
scopic data on cytochrome c oxidase cited above, it was suggested (Karu and Afanasyeva, 1995) that the
820 nm band belongs to the oxidized CuA, the 760 nm band to the reduced CuB, the 680 nm band to
the oxidized CuB, and the 620 band to the reduced CuA (Figure 7.2). The 400–450 nm band is more likely
to be the envelope of a few absorption bands in the range 350–500 nm (i.e., a superposition of several
bands). The band with a maximum near 404–420 nm can be assigned to the oxidized heme, whereas the
longer-wave edge of the envelope at 450 nm (due to its asymmetry), should evidently be assigned to the
reduced CuB. The participation of the heme in the action spectra is confirmed by the optimal dose ratio
(10 and 100 J/m2 respectively for 404 and other visible region maxima, Karu et al., 1984a, b). It should
be noted that the Soret band of heme compounds (i.e., the band in the range 400–420 nm) is more
intense by an order of magnitude than the absorption bands of these compounds in the visible region
(Gouterman, 1978). The weak band at 330 nm may belong to the oxidized CuB. Thus, the bands at 330,
404–420, 680 and 825 nm can be attributed to the oxidized form of cytochrome c oxidase; the edge of
the blue-violet band at 450 nm and the distinct bands at 620 and 760 nm belong to the reduced form
of the enzyme.
Analysis of the band shapes in the action spectra (Figure 7.2) and the line intensity ratios enables us
to conclude that cytochrome c oxidase cannot be considered as a primary photoreceptor when it is fully
oxidized or fully reduced, but only when it is in one of the intermediate forms (partially reduced, or
mixed-valence enzyme).
The conclusion that the action spectra (Figure 7.2) reflect the absorption spectrum of one of the
intermediate forms of the enzyme complex is supported by the results of experiments using dichromatic
irradiation (Figure 7.4). As well as irradiating the cells with light of different wavelengths as normal
(shown on the abscissa), they were simultaneously irradiated with light at 632.8 nm. This wavelength
was close to the position of one maximum in the action spectrum in Figure 7.2 (620 nm). The technique
of this experiment is described in a paper by Karu et al. (1985). The light doses in both irradiation
processes were either optimal (100 J/m2 in the region 600–850 nm, (Figure 7.4, curve 3), and 10 J/m2
(Figure 7.4, curve 1)), or increased (25 J/m2 in the blue-green region, Figure 7.4, curve 2). So, in the case
of simultaneous dichromatic irradiation, a new action spectrum is formed. The comparison of the action
spectra in Figures 7.2 and 7.4. reveals essential differences between them: the shift of the blue-violet
maximum from 404 nm to 450 nm or its absence in the spectrum; new bands in the green region (550–560
nm); the absence of bands at 680, 760, 825 nm (Figure 7.4). It is known (Palmer et al., 1978) that the
shift of the band from 400 to 450 nm can be observed in the course of cytocrome c oxidase reduction.
It should be noted that, in the range 550–560 nm, one can observe an absorption band of one of the
intermediate forms (Brunori and Chance, 1988). These results enable us to conclude that simultaneous
dichromatic irradiation changes the ratio of the reduced and oxidized forms of the enzyme as compared
with ordinary irradiation (Karu et al., 1984a,b).
The suggestion that an intermediate form of cytochrome c oxidase might be the primary photoacceptor
is also supported by the results of Pastore et al. (2000). The fully oxidized form of the enzyme appeared
to be insensitive to He-Ne laser radiation as revealed by absorption spectra. The irradiation increased
the absorption of the partially reduced enzyme as well as its proton pump activity.
It is worth noting that when the cellular monolayer was irradiated simultaneously with λ = 633 nm
and various wavelengths of visible light, the red-far red peaks at 680 and 760 nm disappeared (Figure 7.4).
When the cells were irradiated consecutively with wavelengths 633 and 760 nm, and the time interval
between the two irradiation events was varied, the DNA synthesis rate depended on the order in which
the wavelengths were used (Figure 7.5a). Irradiation first with the light at 760 nm and then with the red
light (λ = 633 nm) stimulated the DNA synthesis, whereas irradiation in the reverse order (633 nm
followed by 760 nm), inhibited it. These effects reached their maxima when the time interval between
the successive irradiation events was between 1 and 3 min and became progressively less pronounced
with a further increase in the interval. It should be noted that the effects were not equal in magnitude:
stimulation amounted to 60%, while inhibition was 20% (Figure 7.5a). This result supports the conclu-
sion that the 620 nm and 760 nm bands do not belong to the one photoacceptor, but to its different
absorbing centers (probably CuA and CuB). Electronic excitation of these centers in a different sequence
may influence the electron transfer in cytchrome c oxidase in a different way, one that has an effect on
the final photobiological response, namely DNA synthesis.
When the consecutive irradiation was performed with red (633 nm) and blue (404 nm) light, the
sequence 633 nm followed by 404 nm had no effect on DNA synthesis, but the sequence 404 nm followed
by 633 nm stimulated it (Figure 7.5b). Again, as in the previous case (Figure 7.4a), the effects did not
occur until the interval between the two irradiation events reached 10 seconds, then increased as the
FIGURE 7.4 Action spectra of simultaneous dichromatic irradiation with λ = 632.8 nm and λ (wavelengths shown
on the abscissa) on the DNA synthesis rate in exponentially growing HeLa cells (adapted from Karu et al., 1985).
FIGURE 7.5 Stimulation of DNA synthesis rate in HeLa cells measured after consecutive irradiation with (a) far
red and red, or (b) blue and red light as dependence of irradiation wavelength sequence and the time interval τ
between the two irradiation events (shown on abscissa) (adapted from Karu et al., 1985). The doses were 100 J/m2
for radiation at 760 and 633 nm, and 10J/m2 for radiation at 404 nm.
interval was increased. The difference between the results of two experiments presented in Figure 7.4a,b
is that in the case of far red-red light-irradiations (Figure 7.5a) the effect disappeared (was reduced to
control levels) when the time interval was increased. In the second case, when irradiation was performed
in the sequence 404 nm + 633 nm (Figure 7.5b), the effect was still maximal at the same time interval
of 104 s. It is quite possible that chromophores absorbing at 404 and 633 nm do not belong to one
molecule but to different molecules of the same redox chain.
Let us recall here three results indicating the antagonistic action of red and far red light. First, when
irradiating isolated rat liver mitochondria, Gordon and Surrey (1960) found that red light at 650 nm
increased oxidative phophorylation but far red light at 725 nm inhibited it. Irradiating hamster
fibroblasts with red (632.8 nm) and far red (760 nm) light caused a change in the intracellular
concentration of cAMP (Karu et al., 1987a), the cAMP concentration behaviors in the former case
being opposite to that in the latter case (increase and decrease, respectively). Irradiation at 670 and
830 nm stimulated the proliferation of the Schwann cells but irradiation at 780 nm inhibited it (van
Breugel et al., 1993). These results could be explained by taking into account the fact that the wave-
lengths mentioned above are absorbed by different chromophores in a different redox state: λmax at
620 nm by CuA (reduced), λmax at 680 nm by CuB (oxidized), λmax at 760 nm by CuB (reduced), and
λmax 820 nm, by CuA (oxidized). Possibly, different absorbing chromophores may play a different role
in driving the metabolism.
One important step in identifying the photoacceptor molecule is to compare the absorption and action
spectra (Hartmann, 1983). Recording an absorption spectrum of a cellular monolayer or individual cell
is not an easy task. The absorption spectra of individual cells were recorded years ago with the aim of
identifying respiratory chain carriers (Nicholls and Elliot, 1974). Absorption spectrum of eight parallel
monolayers of human fibroblasts was recorded using a commercial double beam spectrophotometer (van
Breugel and Bär, 1992). For recording the absorption of one layer with an aim to study the irradiation-
induced changes in absorption of cell chromophores, a sensible multichannel registration method was
developed (Karu et al., 1998, 2000). The first results of these experiments are presented in Figure 7.6 a,b.
FIGURE 7.6 Absorption spectrum of monolayer of dry HeLa cells. The experimental details are described in the
paper by Karu et al. (1998).
First, we recorded the absorption spectra of a dry monolayer (the monolayer was held in air at room
temperature for 30 min. after being rinsed with Hanks’ solution). An example of such a spectrum is
presented in Figure 7.6. The spectrum exhibits distinct absorption bands with maxima at 620 nm, 680
nm (with a shoulder manifest at 665 nm), 810 nm, and 870 nm, weak bands being observed to occur at
715 nm, 730 nm, and 765 nm. Irradiating this type of monolayer with laser radiation had no effect at all.
This circumstance is explained by the fact that virtually not one cell survived the drying, which was
verified through staining with trypan blue.
Further experiments were aimed at recording the absorption spectra of a wet monolayer immediately
after rinsing with Hanks’ solution. To improve sensitivity, the spectra in this series were recorded in the
wavelength range 640–710 nm (585–900 nm in the preceding series). In the given wavelength range
(640–710 nm), there are clearly manifest absorption bands at 670, 718, and 744 nm, and also a less
distinct band or a band shoulder in the vicinity of 750 nm (curves 1 in Figs. 7.7a and b).
FIGURE 7.7 Absorption spectrum of the HeLa monolayer immediately after the removal of the nutrient medium
(curve 1) and following exposure to radiation with λ = 670 nm for the first time (curve 2), second time (curve 3),
and third time (curve 4) (each exposure – 10 s in a dose of 6.3 × 103 J/m2) (Karu et al., 1998); (b): absorption spectrum
of the HeLa monolayer immediately after the removal of the nutrient medium (curve 1) and following exposure to
radiation with λ = 820 nm for the first time (curve 2), second time (curve 3), and third time (curve 4) (each exposure
– 10 s in a dose of 6.3 × 103 J/m2, as described by Karu et al., 1998).
Exposing the sample for 10 s to laser radiation with a wavelength of 670 nm and a dose of 6.3×103
J/m2 once (curve 2 of Figure 7.7a), twice (curve 3 of Figure 7.7b), or thrice (curve 4 of Figure 7.7a) caused
changes in its absorption bands around 670, 750, and 774 nm, the absorption band at 718 nm remaining
unchanged (see Figure 7.7a). In the action spectra, the band in the neighborhood of 670–680 nm belongs
supposedly to the chromophore CuB in the oxidized state, while that in the vicinity of 760–770 nm
belongs to the chromophore CuB in the reduced state (Karu and Afanasyeva, 1995). If there is a corre-
spondence between the action spectra bands (Figure 7.2) and the absorption spectra bands presented in
Figure 7.6, the results presented in Figure 7.7a are quite natural; as laser irradiation increases absorption
in the band at 670 nm, hence the concentration of the chromophore in the oxidized state, absorption
near 750–770 nm (and the concentration of the reduced chromophore) decreases. It is interesting to
compare the responses of the monolayer to the first, second, and third exposure to laser radiation. The
first, with a wavelength of 670 nm, causes no substantial changes in the 670-nm absorption band (curve
2) as compared with that of the unexposed monolayer (curve 1). Only the subsequent two exposures
result in the intensification of the 670-nm (band curves 3 and 4, respectively). An entirely different effect
is observed to occur in the complex absorption band in the region of 745–780 nm. The first exposure
(curve 2) brings about a sharp change in both the intensity and shape of the complex band profile,
practically no changes taking place as a result of the second and third exposures (curves 3 and 4,
respectively), which is very similar to the saturation effect. Two bands, one with λ = 745 and a shoulder
at λ = 755 nm, and the other with λ= 775 nm, are clearly defined in the absorption spectra of the exposed
monolayer (curves 2–4 in Figure 7.7a). The shape of the band points to the presence of two conformers
in the hypothetical reduced chromophore.
The exposure of the cellular monolayer to laser light with λ = 820 nm (Figure 7.7b) was also observed
to cause changes in the absorption bands in the vicinity of 670 and 775 nm. Recall that the action spectra
featured a band at around 825 nm, which is supposedly associated with the oxidized chromophore CuA
(Figure 7.2). The exposure of the monolayer to light with λ = 820 nm was carried out in the same way
as in the case of laser light with λ= 670 nm in the preceding series of experiments. The sample was
exposed for 10 s to the radiation in a dose of 6.3 × 103 J/m2 once (curve 2), twice (curve 3), or thrice
(curve 4). Following the first exposure (curve 2), a sharp increase of absorption is observed to occur in
the band near 670 nm (and the correspondingly sharp reduction of absorption in the band near 770
nm) in comparison with the intact monolayer (curve 1). The second (curve 3) and the third (curve 4)
exposure cause no sharp changes in absorption, which is likely due to an equilibrium being established
between the oxidized and reduced forms of the chromophore CuB.
Clearly manifest in Figure 7.7b (curves 2–4) are two bands, one with λ1 = 750 nm and the other with
λ2 = 770 nm, as was also in the case with irradiation at λ = 670 nm (Figure 7.7a). One can therefore
state with certain reservations that radiation with a wavelength of 670 nm or 820 nm has most likely no
effect on the chromophore reduction mechanism. In both cases (λ = 670 nm and λ = 820 nm), irradiation
reduces the absorption of radiation and the concentration of the reduced form of the chromophore, and
gives rise to its two conformably ordered forms.
On the contrary, the behavior of the 670-nm band in the case of exposure to laser light with λ = 820 nm
(Figure 7.7b) differs drastically from that in Figure 7.7a (i.e., in the case of irradiation at λ = 670 nm). The
very first exposure to light with λ = 820 nm increases the intensity of the 670-nm band (curve 2 of Figure 7.7b).
Following the second exposure (curve 3), this band changes but little, and practically no changes are observed
to occur following the third exposure (curve 4) (there takes place something like saturation).
Thus, the laser wavelength and the number of exposures have different (opposite) effects on the
(supposedly) oxidized form of the chromophore, with the absorption maximum at λ = 670 nm. Radiation
with λ = 820 nm oxidizes the chromophore CuB stronger in the first exposure (i.e., oxidation proceeds
in a stepwise manner), whereas radiation with λ = 670 nm has practically no effect on the oxidized form
of CuB in the first exposure (Figure 7.7a). It is only after the second and the third exposure that the
concentration of the oxidized form of the chromophore grows higher, no saturation being reached in
our experiments.
The 718-nm band suffers virtually no changes following exposure to laser light differing in wavelength.
It should be noted that this band is not manifest in the action spectra (Figure 7.2). The absence of changes
in absorption following exposure to radiation with two wavelengths (Figure 7.7a, b) also seems quite
logical and gives reason to believe that the chromophore absorbing in this region takes no part in the
photoregulation process responsible for the action spectrum.
The first results of the experiments on irradiating the cellular monolayer bear witness to the fact that
the method developed (Karu et al., 1998) allows one to measure the weak absorption of a live cellular
monolayer and holds much promise for detailed research into the primary changes in the photoacceptor
molecule (cytochrome c oxidase) consecutive upon the absorption of light by its various chromophores
(CuA and CuB, for example). New results of this type of measurement are presented in the paper of Karu
et al., 2001.
7.2.2 Primary Mechanisms of Light Action

The primary mechanisms of light action on the photoacceptor molecules have not yet been established.
Below, four main possibilities discussed in the literature so far are considered. It has been suggested that
one possible process involves an acceleration of electron transfer in the respiratory chain due to a change
in the redox properties of the carriers following photoexcitation of their electronic states (Karu, 1988; 1989a).
A similar principle governs the function of photosynthetic reaction centers (Govindjee, 1982) and is involved
in many so-called “blue light responses” (Senger, 1980, 1984). It is well known that electron excitation
changes the redox properties of molecules (Marcus and Sutin, 1985). The results of the recent measurements
(Karu et al., 1998, 2000; Figure 7.7), as well as the discussion from Section 7.2.1, allow one to propose that
the photoexcitation of certain reaction centers in cytochrome c oxidase molecule (like CuA and CuB or hemes
a and a3) or in cyt bd and bo complexes of Escherichia coli (Afanasyeva et al., 1995) influences the redox
state of these centers, and consequently, the rate of the electron flow in the molecule. It was suggested, for
example, that irradiation shifts the cytochromes to more reduced forms that naturally promote electron
transport (Adar and Yonetani, 1978). The laser irradiation and activation of electron flow in the molecule
of cytochrome c oxidase can reverse the partial inhibition of its catalytic center by NO (Karu, Tyatibrat,
Kalendo, 2001). Recently, the redox absorbance changes of the respiratory chain components of Escherichia
coli were measured following He-Ne laser irradiation. A dose of 4.3 × 104 J/m2 led to partial oxidation of
cyt b and cyt d, while flavoproteins were found to be slightly reduced (Dube et al., 1997).
During light excitation of electronic states, a noticeable fraction of the excitation energy is inevitably
converted to heat, which causes a local increase in the temperature of the absorbing chromophores
(Letokhov, 1991). Any appreciable time- or space-averaged heating of the sample can be prevented by
controlling the irradiation intensity and dose appropriately. However, there is still the possibility of
localized transient heating of absorbing chromophores. The local transient rise in temperature of absorbing
biomolecules may cause structural (e.g., conformational) changes and trigger biochemical activity (sec-
ondary dark reactions) such as activation or inhibition of enzymes.
To evaluate the contribution of local transient heating of light-absorbing microregions to biochemical
activity, Escherichia coli (Karu et al., 1991a) and HeLa cells (Karu, 1992a) were irradiated using femtosecond
laser pulses (τ = 620 nm, τp = 3 × 10-13 s, f = 0.5 Hz, Ep = 1.1 × 10-3 J/cm2, Iav =5.5 × 10-4 W/cm2, Ip = 109
W/cm2), as well as CW laser radiation (λ = 632.8 nm, I = 1.3 W/cm2). The irradiation dose required to
produce a similar biological effect (an increase in the clonogenic activity of irradiated cells compared with
the non-irradiated control) was found to be a factor of about 102–103 lower for pulsed radiation than for
CW radiation. The minimum size of the microregions transiently heated by irradiation with femtosecond
laser pulses was estimated at about 10 Å, which corresponds to the size of the chromophores of the
hypothetical primary photoacceptors — the respiratory chain components (Karu et al., 1991a).
The effects of slight local heating and a substantial local temperature gradient (Letokhov, 1991) occur
for both CW and femtosecond pulses. The difference is that the density of such local heating sites in the
case of CW radiation is extremely low because of the low radiation intensity. This density is lower than
the density of the absorbing chromophores by a factor of approximately 107–1010, depending on the
relaxation time and excitation cross section of the chromophores. In the case of femtosecond pulses, the
chromophores become excited, which causes a stronger biological response.
Therefore, it can be concluded that higher than average local transient heating of microregions with
a size characteristic of absorbing chromophores is possible. The evaluation of the transient heat intensity
of these microregions and the diffusion of heat from them requires a more detailed analysis. One should
note here that the above considerations involved individual cells. The problem of local heating of
absorbing chromophores takes entirely new dimensions and significance when whole tissues are irradi-
ated. Some considerations of this problem are found in papers by Letokhov (1991), Karu et al. (1997),
and Karu (2002).
The principal use of oxygen in a respiratory chain involves its 4-electron reduction to water. In normal
metabolic processes, as well as in a number of nonenzymatic biological reactions, both univalent and
Ca2+ Uptake or ATP Production
NADH Q cyt b cyt c1 cyt c cyt a/a3 H2 O
Transhydrogenase
SOD Catalase
Q2- H2 O2
R
ROOH
GSH
GSPX
GSH Reductase
NADPH
Ca2+ Release
or Uncoupling
FIGURE 7.8 The connection of antioxidant enzymes to electron transport, oxidative phosphorylation and Ca2+
flux. Abbreviations: GSH – glutathione; GSPx – glutathione peroxidase; SOD – superoxide dismutase; Cat – catalase;
R – reduced lipid; ROOM – lipid peroxide (after Forman and Boveris, 1982).
bivalent reductions of molecular oxygen also occur (Figure 7.8). For example, free radicals like
HO.(hydroxyl) and O2•− (superoxide) appear as a result of 1-electron reduction. It has been shown that,
in mitochondrial electron transport, the superoxide radical is produced, and the production of O2•, as
well as the product of its dismutation, H2O2, primarily depend on the metabolic state of the mitochondria
(Forman and Boveris, 1982). By activating electron flow in the respiratory chain, one can also expect
increasing O2•− production. Some data in the literature also suggest that, besides the above-mentioned
method of generating O2•−, there is another NADH-related way to generate O2•− in the mitochondria
(Nohl, 1987).
It has been demonstrated experimentally that mitochondria possess a mechanism for the reabsorption
of O2•−, and O2•− may be a source of electrons for the oxydative phosphorylation of ADP under physio-
logical conditions (Mailer, 1990). Experimental data show that liver mitochondrial ATP synthesis can be
inhibited and promoted by the UV generation of O2•− (Dmitriev et al., 1990). Recall also that even a
small increase in O2•− concentration in a cell results in multiple responses like increase in [Ca2+]i and
pHi (alkalization), release of arachidonate, activation of Na+/H+ antiport and Ca2+ ATPase, alteration of
Na+ – Ca2+ exchange (Murphy et al., 1988; Shibanuma et al., 1988; Kaneko et al., 1990).
Experiments measuring the luminol-amplified chemiluminescence of murine splenocytes after near
IR irradiation (Karu et al., 1993b) do not exclude the possibility of increased O2•− concentration. A
comparison of the action spectrum of chemiluminescence stimulated after irradiation with various
wavelengths of light and the absorption spectrum of the cyt c oxidase indicated some similarity between
these two. As the absorption band of cyt a/a3 at 830 nm is thought to be due to its copper component,
and the chemiluminescence emitted by mitochondria is believed to be copper-dependent, one should
consider the possibility that some low-power laser effects can be related to increased O2•− production.
This possibility, discussed in a paper of Karu et al. (1993b), becomes more serious if we take into account
the fact that H2O2, the product of O2•− dismutation, is involved in a complex set of reactions linking the
H2O2 production in mitochondria to the regulation of cellular metabolism (Forman and Boveris, 1982).
As shown in this reference and also in Figure 7.8, many antioxidant enzymes are involved in this link.
One of the enzymes taking part in the pathway is catalase. Catalase has been shown to be involved in
the increase in protein synthesis induced by He-Ne laser light in yeast cells (Karu et al., 1993c). In that
study, the activity of catalase was measured immediately after the irradiation of Torulopsis sphaerica and
the amount of synthesized protein was tested 18 h later. The activity of catalase as well as the amount of
synthesized protein were increased in the irradiated cells. The inhibition of catalase activity by 3-amino-
1,2,4-triazole at the moment of irradiation suppressed protein synthesis. At the same time, the protein
synthesis was not affected by 3-amino-1,2,4-triazole in nonirradiated cells. Possible specific links between
the increase in catalase activity and protein synthesis in irradiated cells were discussed in the paper of
Karu et al. (1993c).
Certain photoabsorbing molecules like porphyrins and flavoproteins (some respiratory chain com-
ponents belong to these classes of compounds) can be reversibly converted to photosensitizers (Spikes,
1989; Giese, 1980). Based on monochromatic visible light action spectra for DNA synthesis in HeLa
cells, and spectroscopic data for porphyrins and flavins, a hypothesis was advanced that the absorption
of light quanta by these molecules is responsible for the generation of singlet oxygen, 1O2 (Karu et al.,
Kalendo1984a). Chemically active 1∆g and ∆+gstates of oxygen with energies of 1.0 and 1.5 eV might
play an active mediator role in achieving the biological effects of irradiation. This possibility has been
considered for some time as a predominant reaction during irradiation of cells with high doses and
intensities of light (Karu, 1988; 1989b). The possible role of singlet oxygen in low-power laser effects
was discussed in several other papers (Danilov et al., 1990; Friedman et al., 1991). However, experi-
ments demonstrating the generation of singlet oxygen in cells or tissues after irradiation as well as
examination of the possible secondary (dark) biochemical reactions are to date nonexistent. Figure 7.9
presents two principal pathways for 1O2 generation, as discussed in the papers cited above. The pathway
on the left includes strong permitted transitions and, for this reason, is highly probable (classical
photodynamic mechanism). This pathway was discussed in papers by Karu, 1988; Karu et al., 1984a;
Friedman et al., 1991. In contrast, the pathway on the right, showing direct excitation of the oxygen
molecule, which was discussed in the paper by Danilov et al. (1990), has extremely low probability
due to forbidden transitions.
The light effects of respiration are oxygen-dependent and prevented by anaerobiosis; it is generally
believed that photodynamic reactions promoted by certain respiratory chain components (flavins, hemes
and Fe-S centers) are associated and occur in aerobic conditions only (Epel, 1973; Giese, 1980; Spikes,
1989; Kim and Jung, 1995). Irradiating yeasts in aerobic and anaerobic conditions with an He-Ne laser
increased protein synthesis (which was measured as the final photobiological macroeffect) in both cases,
the only difference being the dose range (Karu et al., 1993d). This finding indicates that, at least in this
particular case, the 1O2-connected mechanism is not involved.
FIGURE 7.9 Two principal ways for generation of singlet oxygen in a cell: (a) photodynamic action; (b) direct
excitation of triplet oxygen. Details can be found in the text.
Taken together, there is certainly more than one reaction involved in the primary mechanisms of low-
power laser effects. The various possible reactions discussed above are summarized in Figure 7.10. There
are no grounds to believe that only one of these processes occurs when a cell is irradiated. An important
question for the future is which of these reactions is responsible for a certain low-power laser effect.
However, recent experimental results of measurements of redox absorbance changes of living cells after
the irradiation (Dube et al., 1997; Karu et al., 1998, 2000) clearly indicate that mechanism based on
changes in redox properties of terminal enzymes of respiratory chains might be crucial.
changes in redox properties and

acceleration of electron transfer
O2 -
S1 O2
O2 one-electron auto-oxidation
1
O2
photodynamic action
hv Thermal
relaxation
changes in biochemical activity
induced by local transient
heating of chromophores
S0
FIGURE 7.10 Possible primary reactions with a photoacceptor molecule after promotion to excited electronic states.
This theoretical scheme does not mean that all relevant reactions occur from the first singlet state.
7.2.3 Secondary Reactions of Light Action

Sections 7.2.1 and 7.2.2 considered photoacceptor molecules and possible primary reactions occurring
under irradiation. On the other hand, it is well known that many biochemical reactions in cells occur
hours and even days after the irradiation procedure. Usually, the irradiation lasts in a timescale of seconds
and minutes. The biological responses of cells occurring later, when the radiation is switched off, are
called secondary reactions. The specificity of light action is believed to be due to the quanta absorption
by a photoacceptor, cytochrome c oxidase molecule (Section 7.2.1). Also, a flavoprotein like NADH-
dehydrogenase has been discussed as a possible photoacceptor for blue and red light (Karu, 1988, 1989a).
If the photoacceptors are located in the mitochondria (Section 7.2.2), then how are the primary
reactions occurring in the respiratory chain (Section 7.2.2) connected with DNA synthesis in the nucleus?
A scheme presented in Figure 7.11b can explain this. This scheme is based on the fact that a redox chain
such as the respiratory chain is capable of controlling cellular homeostasis. The photoexcitation induces
changes in cyt a/a3 (Section 7.2.2) or in flavinic components of the chain (e.g., NADH-dehydrogenase,
Karu, 1988, 1989a); this event can, in turn, cause other redox changes and modulations of biochemical
reactions through a photosignal transduction and amplification chain, which leads to a photobiological
macroeffect, such as increased proliferation (on Figure 7.11b,c marked by DNA synthesis).
The absorption of light quanta by the respiratory chain components causes a short-term activation
of the respiratory chain and oxidation of the NADH pool. It is known that oxidation of the NADH
pool leads to changes in the redox state of both mitochondria and cytoplasm (Krebs and Veech, 1970;
Kohen et al., 1983). The activation of the electron transport chain also results in an increase with
proton motive force (pmf, ∆µH+), electrical potential of mitochondrial membrane (∆ψ), ATP pool,
and acidification of the cytoplasm. It has been confirmed experimentally that this is also true in the
case of respiratory chain activation by irradiation: changes in pmf, ∆ψ, and ∆pH as well as extrasyn-
thesis of ATP have been achieved by irradiating mitochondria with a He-Ne laser (Passarella et al.,
1984). By irradiating cells with a wideband visible light of λ > 400 nm, the enhancement of the activity
of ATP-synthase was observed (Nedelina et al., 1985). The ATP level was also found to be raised after
irradiation of whole cells: human lymphocytes at 820 nm (Herbert et al., 1989), R3230AC adenocar-
cinoma cells at 630 nm (Hilf et al., 1986), HeLa cells at 632.8 nm (Karu et al., 1995a). Irradiating
human lymphocytes with a He-Ne laser also caused changes in the ultrastructure of mitochondria,
such as the formation of giant mitochondria, which was interpreted by the authors as an intensification
of the energy metabolism (Bakeeva et al., 1993). A perturbation of the mitochondrial energy production
was measured by rhodamine 123 uptake, when human oral fibroblasts were irradiated at 812 nm
(Loevschall and Arenholt-Bindslev, 1994a).
FIGURE 7.11 A possible mechanism of light-enhanced proliferation of mammalian cells. Monochromatic visible
and near IR radiation initially absorbed by mitochondria (a) eventually causes stimulation of DNA synthesis (c) after
numerous intervening dark reactions (b). Eh ↑-shift of cellular redox potential to moe oxidied direction; the arrows
↑ and ↓ indicate increase or decrease of respective values, the brackets[] indicate the intracellular concentration of
respective chemicals.
The acidification of the cytoplasm (rise of intracellular H+ concentration), caused by the activation of
the respiratory chain, controls allosterically the activity of the Na+/H+ antiporter situated in the cyto-
plasmic membrane (Pouyssegur et al., 1985). This enzyme plays a key part in the alkalization of the
intracellular medium. A short-term increase in the intracellular pH (∆pHi) is one of the necessary
components involved in the transmission of mitogenic signals in the cell (Hesketh et al., 1985, Moolenaar,
1986; Pouyssegur et al., 1985).
A change in pHi (alkalization of the cytoplasm after irradiation) has been measured experimentally.
The pH of rat brain was measured during 1 week following photodynamic therapy (Chopp et al., 1990).
In a control (light only) experiment, the tissue pH was elevated after irradiation at 70 and 140 J/m2, as
compared with nonirradiated control (∆pH = 0.2–0.3 units).
In an eukaryotic cell, a change in the redox state of the mitochondria causes changes in the redox state
of the cytoplasm or, in other words, changes the overall redox state of a cell. In Figure 7.11b, this event
is marked by the NAD/NADH couple. More exactly, the overall redox state of the cell represents the net
balance between stable and unstable reducing and oxidizing equivalents in dynamic equilibrium and is
determined by three couples: NAD/NADH, NADP/NADPH, and GSH/GSSG (GSH = glutathione). These
pairs are mutually dependent, and altering the ratio in one couple causes changes in the others. Intrac-
ellular pH (pHi) is also closely connected to these ratios.
Experiments described in papers by Keyse and Tyrrell (1987, 1989) support the suggestion of the
crucial role of redox changes in alterations of cellular metabolism. It has been demonstrated that agents
that reduce the level of available glutathione in human skin fibroblasts, i.e.. that shift the cellular redox
state to a more oxidized direction, also induce heme oxygenase. In other words, it appears that the level
of the heme oxygenase is fully regulated by the redox state of the cell (Tyrrell, 1992). Among the agents
causing this effect were UVA radiation, visible light at 405 nm, hydrogen peroxide, cadmium chloride,
iodoacetamine, and menadione (Keyse and Tyrrell, 1987, 1989).
The importance of this finding is as follows: heme oxygenase appears to be a 32-kDa stress protein.
Stress proteins are synthesized in cells as a response to the action of a wide variety of chemical and
physical factors. The most studied stress proteins are the heat shock proteins (Schlesinger et al., 1982).
Factors such as glucose deprivation and hypoxic stress induce the synthesis of stress proteins unrelated
to the heat shock proteins, and the 32-kDa stress protein is also believed to be distinct (Keyse and Tyrrell
1989). Keyse and Tyrrell (1989) have proposed that the induction of heme oxygenase may be a general
response to an oxidant stress. Modulation of cellular redox state affects gene expression via mechanisms
of cellular signalling e.g., NF – κ-B and AP-1 (Figure 7.11b).
There is also an indirect indicator that He-Ne laser irradiation alters the redox state of cells. It is known
that any shift in the redox state and metabolic activity of a cell will necessarily alter its radiosensitivity,
adaptive response to radiation or other forms of endogeneous on exogeneous free radical toxicity (Green-
stock, 1986). In experiments when the γ-irradiated monolayer of HeLa cells was irradiated with a He-
Ne laser at various time intervals before exposure to ionizing radiation, the viability of the cells previously
irradiated with a laser increased significantly as compared with γ-irradiated cells (Karu et al., 1994a).
The Na+/H+ antiporter is not the only cell membrane enzyme to participate actively in photosignal
transduction and amplification chain. Other ion carriers such as Na+, K+-ATPase and enzymes controlling
cAMP level in a cell are also activated. The activation of the Na+, K+-ATPase following He-Ne laser
irradiation of human erythrocytes (Moroz, 1983) and diode laser (λ = 830 nm) irradiation on a rat
saphenous nerve (Kudoh et al., 1989) has been established. Experimental data on changes in cellular
cAMP level after irradiation with light of various wavelengths provide reason to believe that the action
of light on proliferation may be connected with the regulation of cell metabolism via cAMP (Karu et al.,
1987a).
Far more than one cellular response to irradiation are connected with the plasma membrane. The
depolarization of spontaneously active neurons in subesophageal ganglia of Helix pomatia occurred after
irradiation with a He-Ne laser (Balaban et al., 1992). The intracellular Ca+ concentration was found to
rise during the first few minutes after He-Ne laser irradiation of human lymphocytes (Karu et al., 1991b)
and neutrophils (Loevschall et al., 1994c) as well as bovine sperm cells (Lubart et al., 1997) and rat
Schwann cells (van Breugel et al., 1993). Depending on the time elapsed after irradiation and the
wavelength used. Irradiation increased both the cell–cell and cell–glass adhesion (Karu et al., 1996a).
Direct measurement of ionic currents through the plasma membrane of both excitable (cardiomyocytes,
neurons) and nonexcitable (glial) cells using the patch-clamp technique under He-Ne laser irradiation
showed the activation of background channels, probably ATP-dependent K+-channels or Ca+-dependent
K+-channels (Karu et al., 1996b). First, the ATP-dependence of the light-sensitive background single
channel currents supports the scheme of photosignal transduction chain under discussion (Figure 7.11b).
Furthermore, in the same series of experiments, it was established that the light-sensitive ion currents
were recorded only in a cell-attached configuration of the patch pipette, i.e., in conditions of cellular
integrity, but not in a whole-cell configuration. Indeed, the cascade of biochemical reactions (photosignal
transduction and amplification chain) depends on the cellular homeostasis and can occur only in
conditions of cellular integrity.
The photosignal transduction and amplification chain in its part from plasma membrane to nucleus
is not specific for light signal but includes also a standard way of controlling cell proliferation (cAMP
level, changes in intracellular contentation of H+, K+, Na+, Ca2+). This is a complicated area in cell biology
and the regulation mechanisms are not fully clear. The interested reader is referred to papers of Boyton
and Whitfield (1983), Cone (1971), Kaplan (1978), Rozengurt (1986), Rozengurt and Mendoza (1980),
Hesketh et al. (1985), Hülser and Frank (1971), Moolenaar (1986), Pouyssegur et al. (1985). The alter-
nation of the cellular homeostasis parameters leads to a parallel shift of different reactions, and it is not
easy to establish the causal relationships.
In Figure 7.11b, two principal regulation paths by light were suggested. The first is the existence of a
connection between the light-activated redox functions of mitochondria, changes in redox state of
cytoplasm, activation of certain redox sensitive transcription factors, the depolarization of cellular mem-
brane, and the rise of intracellular pH (alkalization of cytoplasm). The second is the photoacceptor
control over the level of intracellular ATP. As we know, even small changes in ATP level can alter the
cellular metabolism significantly (Brown, 1992).
7.3 Explanation of Controversies and Limitations of Low-Power

Laser Effects on Cellular Level
Despite almost 30 years of clinical use, until now, a considerable degree of scepticism concerning the use
and clinical efficacy of low-power lasers existed. This is, in part, a consequence of the generally poor
quality of many original publications, as well as the lack of critical scientific scrutiny of the many claims
made for these devices. In recent years, this situation has begun to change. Many high-quality publications
within the medical and scientific literature attest to the clinical usefulness of light sources (lasers and
LEDs) and quantify their photobiological effects in the laboratory.
Years ago, the usual perception was that laser biostimulation occurred in Eastern laboratories but not
in Western. At that time, the actual situation was indeed rather close to this. Very few Western scientists
took low-power laser effects seriously and even fewer performed experiments. There have been changes
in this attitude recently, and a number of well-designed experiments have been performed on various
cells. The reader will find references to these throughout this book. In some cases, the effects of visible
and near IR light on various cellular functions have been described in papers that were not dealing with
the issue of low-intensity laser effects at all (e.g., Kato et al., 1981; Marchesini et al., 1989; Chopp et al.,
1990; Albrecht-Büehler, 1991). This gives a greater reliability to those results for the field of low-power
laser effects.
Based on the literature data gathered so far on the cellular level, the main controversies and limitations
will be analyzed in this chapter. Some that are no longer topical were mentioned in the Introduction.
7.3.1 Magnitude of Low-Power Laser Effects

One confusing point about low-power laser effects has been their possible degree (a strong effect, a weak
effect, or no effect at all). Indeed, even when the same culture of cells is irradiated, the degree of biological
response can differ. Based on the discussion and data from Section 7.2, one can clarify this issue with
the aid of Figure 7.12. It was concluded in Section 7.2.3 that one key event among the secondary reactions
of cellular responses was the change in overall redox state of the irradiated cell. Recall that the overall
redox state represents the net balance between stable and unstable reducing and oxidizing equivalents in
dynamic equilibrium. The main idea of the diagram in Figure 7.12 is that the cellular response is weak
or absent when the overall redox potential of a cell is optimal or near optimal for the particular growth
conditions, and stronger when the redox potential of the target cell is initially shifted to a more reduced
state (and intracellular pHi is lowered commensurably). This explains why the degrees of cellular response
can differ markedly in different experiments, and can sometimes be nonexistent.
This suggestion is supported by the following experimental results. In two sets of experiments, the
cellular redox state was changed artificially before irradiation. In the first case, the redox state of the HeLa
cells was lowered (shifted into a more reduced direction) by oxygen–nitrogen transition. By such a
transition, the NADH fluorescence increases, which means a shift to the reduced direction (Chance et
al., 1962; upper corner of Figure 7.13). When these cells were irradiated with light of various wavelengths
marked on the abscissa of Figure 7.13, the new action spectrum appeared to be quite different from the
action spectrum in conditions of normal O2 partial pressure. As seen in Figure 7.13, the stimulative effect
of light at 620–630 nm on DNA synthesis is stronger than in normal cultivation conditions. A new peak
appears at λ = 570 nm.
FIGURE 7.12 Schematic illustration of the action of monochromatic visible and near IR radiation on a cell. The
magnitude of the irradiation effect is determined by redox potential of the cell at the moment of irradiation.
TABLE 7.1 Changes in DNA Synthesis Rate in Exponentially Growing HeLa Cells
1.5 h after Irradiation with He-Ne Laser in Various Doses with or without the Presence
of 1 mM Methylene Blue (MB) (Modified from Karu, 1989a)
Dose, J/m2 0 6 30 102 103
Irradiated Cells
MB absent — 102 ± 5% 150 ± 8% 183 ± 12% 153 ± 9%
MB present — 200 ± 9% 227 ± 11% 213 ± 8% 189 ± 6%
Nonirradiated Cells
MB absent 100% — — — —
MB present 202 ± 6% — — — —
In the second set of experiments, the redox state of HeLa cells was shifted before irradiation to oxidized
direction using methylene blue solution. Methylene blue participates readily in a number of oxidation-
reduction reactions as an electron acceptor modulating the intracellular redox state. This supravital dye
has been used as an anti-inflammatory and antibacterial agent, as an antidote for cyanide and carbon
monooxide poisoning, and also as a radio-protectant. It has been shown that methylene blue affects the
concentration of intracellular reducing agents (Hrushesky et al., 1985) and markedly increases oxygen
consumption of fresh tissue homogenates in the presence of adequate NAD(P)H (Hrushesky et al., 1988).
As seen in Table 7.1, irradiating cells pretreated with methylene blue with a He-Ne laser stimulated
the DNA synthesis only a little (a maximum of 25% at a dose of 30 J/m2 ) because the control level
was already elevated twice before the irradiation (202% as compared with nontreated control, 100%)
(Karu, 1989a).
During in vitro measurements of superoxide dismutase and catalase activity after He-Ne laser irradi-
ation (both enzymes have absorption bands near 633 nm), no changes were detected when the enzyme
solutions had a pH corresponding to maximal catalytic activity. In contrast, very pronounced photore-
FIGURE 7.13 Action of visible monochromatic light on DNA synthesis in HeLa cells in normal cultivation conditions
and after 60 s O2/N2 transition. DNA synthesis rate was measured 2.5 h after irradiation of a dose of 100 J/m2 as
described by Karu (1989). In the upper corner: a microfluorimetric recording of the kinetics of fluorescence observed
in oxygen-nitrogen transition of kidney cells (adapted from the paper of Chance et al., 1962).
activation effects were detected when the enzymes were irradiated under non optimal lowered pH
conditions (Zubkova, 1978; Gorbatenkova et al., 1988).
Recall also that a jump in intracellular pH (pHi) after irradiation has been measured experimentally.
pHi is a parameter of cellular homeostasis, which is closely connected with cellular redox state. These
experiments proved that pHi was increased by 0.20 units in mammalian cells due to irradiation with red
light (Chopp et al., 1990) and 0.32 units in E. coli (Quickenden et al., 1995). These two works concluded
that, by means of irradiation, it is indeed possible to shift the pHi and the overall redox state of cells into
a more oxidized direction, as was suggested in Figure 7.12.
Let us now see if this working hypothesis for cellular phenomena can be applied to clinical low-power
laser effects as well. Under normal conditions, mammalian cells maintain a very precise pHi, usually
between 7.0 and 7.2 (Roos and Boron, 1981). The normal pH of arterial blood is 7.4 and healthy tissues
usually have pH values between 7.0 and 7.4 (Vaupel, 1977). Growth factors, neurotransmitters, and direct
cell–cell interactions can modify the regulation of pHi in receptive cells (Roos and Boron, 1981).
The two most important areas of low-power laser therapy are wound healing and treatment of chronic
inflammations. Both these conditions are characterized by decreased oxygen tension (decreased pO2,
hypoxia) and acidosis (decreased pH) (Kittlick, 1986). Most normal tissues have a pO2 about 40 mm Hg,
while, in hypoxic states, the pO2 is in the range of 0–5 mm Hg (Vaupel, 1977; Freitas, 1991). In the case
of normal regeneration, wound hypoxia is a transient condition eventually replaced by a normal pO2
during the final stages of regeneration. The situation is different with chronic injuries. Chronic inflam-
mation is characterized by continued aerobic glycolysis and by a redox shift (measured as the
NAD+/NADH ratio) toward a reduced state. The pO2 of rheumatoid synovial fluid, for example, was
found to fall as low as 0 mm Hg (Kittlick, 1986). As also noted by Kittlick (1986), local hypoxia is
considered to be one of the primary causes of rheumatism.
When irradiating fresh wounds, the effect of irradiation can be minimal or nonexistent. This
happens in cases where the proliferation is active and the regeneration of tissue integrity occurs at a
more or less maximal (normal) rate. This may be the reason there is often no phototherapeutic effect
observed when irradiating fresh experimental wounds. This problem was discussed earlier (Karu,
1987, 1989). Two examples illustrate this conclusion: He-Ne laser irradiation failed to accelerate
healing of fresh wounds in two rat models (Allendorf et al., 1997). Laser radiation at 630 nm
significantly increased the healing of chronic wounds of genetically diabetic mice (Yu et al., 1997).
It is known that intracellular pH affects the contractile function of the heart, its metabolic reactions,
ion exchange and calcium homeostasis. A fall in extracellular pH, whatever the mechanism, causes
a decrease in the heart’s ability to contract, as occurs, for example, in ischemia (Poole-Wilson, 1989).
It should be noted here that low-power lasers are specifically used in treating ischemia (Kashuba,
1981; Korochkin, 1988).
The working hypothesis that an alteration of the intracellular redox state plays the crucial role in low-
power laser effects may have a broader significance than simply that of explaining the laser action on
wound healing and inflammation. When a pharmacological agent interacts with a receptor, usually
situated on the outer cell membrane, it sets in motion a chain of events known as a stimu-
lus–response–recovery cycle. These processes are complicated and consist of many biochemical reactions,
not to mention a multitude of agonists and receptors. There is, however, a common step involved in the
stimulus–response–recovery cycle: a change in the redox state and pHi. Analyzing the data from the
extensive review by Roth et al. (1983), one can see that this phenomenon is involved in almost every
branch of pharmacology.
The working hypothesis introduced above can be extended as follows: the irradiation-altered redox
state of cells modulates their response to biochemical agonists. To illustrate how sensitive and diverse
cellular responses to changes in the cellular redox state may be, refer to Puppi et al. (1968). The
authors of that study artificially altered the redox state in their tissue models (frog ileum and frog
heart) and then studied the action of acetylcholine and adrenaline. Oxidation with methylene blue
caused acetylcholine to act in a stimulatory manner, but, after reduction with ascorbate, acted in an
inhibitory fashion. In contrast, adrenaline was stimulative in oxidizing states, and inhibitive under
reducing conditions.
An attempt was made to quantify the magnitude of the irradiation effect as a dependence on the
metabolic status of the cells at the moment of exposure (Tiphlova and Karu, 1991b). The E. coli culture
was cultivated using various nutrient conditions; minimal growth medium M9 supplemented with
glucose, glycerol, or arabinose (Figure 7.14b,c,d) or Hottinguer broth, (Figure 7.14a).
The kinetics of growth curves of intact cells appeared to be quite different in different conditions. The
population grown with glucose (Figure 7.14b, curve 1) started dividing almost without a latent period.
The specific rate of the exponential growth was equal to 0.55h-1. Irradiating this culture caused an
increased specific rate of exponential growth k = 0.78 h–1 (Figure 7.14b, curve 2). Here k is determined
from Xt = X0ekt; X0 denotes the number of cells in the sample at the beginning of the exponential phase
of growth and Xt, denotes the number after t hours of growth (Andersen and von Meyenburg, 1980).
The difference in the number of cells in the exposed and unexposed cultures after an incubation period
of 60 min was 126% (Figure 7.14b).
In the nutrient medium with glycerol, the control culture had a latent period of about 15 min, and
the specific rate of exponential growth was 0.64 h–1 (Figure 7.14c, curve 1). In an irradiated culture, the
lag-period was almost fully reduced and the specific rate of growth was increased (k = 0.80 h–1). The
difference in the number of cells in the exposed and unexposed cultures at 1-h incubation was 130%
(Figure 7.14c).
FIGURE 7.14 Growth curves of cultures of E. coli WP2 (1) control and (2) irradiated with a He-Ne laser at a dose
of 4 × 103 J/m2, cultivated in (a) Hottinguer broth or M9 medium supplemented with (b) glucose, (c) glucerol, and
(d) L-arabinose (adapted from Tiphlova and Karu, 1991b). Growth stimulation is measured as the ratio of number
of irradiated to control cells after 1 h incubation (in %).
When arabinose was used as the carbon source, the latent period of unexposed culture was longer
than 1 h, and the specific growth rate was 0.74 h-1 (Figure 7.14d, curve 1). The effect of irradiation in
this case manifested itself first of all in the almost complete disappearance of the lag period and an abrupt
increase in the number of cells throughout the first hour of incubation. For this curve, k = 0.80 h-1, and
the difference in the number of cells in the exposed and unexposed cultures came to 145% (Figure 7.14d).
A comparison of curves 1 and 2 in Figure 7.14 makes it possible to reveal the characteristic features
of influence of irradiation with red light on E. coli cultures under different conditions. The difference
between the number of cells in exposed and unexposed cultures is small for cultures with a short latent
period (Figure 7.14b, c) and large if the control culture begins to divide after a long lag period
(Figure 7.14d, as well as Figure 7.14a). The specific rate of exponential growth of exposed culture is almost
the same in all cases (k = 0.78–0.80 h-1). These results show that irradiation under these specific exper-
imental conditions provides the same maximal k for a bacterial culture no matter what the parameters
of the growth curve were. This is a biological limit on growth stimulation.
The effect of irradiation, however, shows up for just a short time, being maximal during the first 45
to 60 min of incubation. Later (2 to 4 h after the start of incubation), there is no difference in the number
of cells in exposed and control cultures. This regulatory mechanism is characteristic of all models studied
(Figure 7.14).
The experimental results obtained indicate that the magnitude of photostimulation of E. coli division
depends on the metabolic state of the initial culture or, in other words, on the presence and duration of
the latent period. On the other hand, there is a maximal specific rate of exponential growth (in our
experimental conditions, 0.80 h–1 ), which limits the degree of photostimulation effect.
7.3.2 Diversity of Low-Power Laser Effects

One major controversy surrounding low-power laser effects has always been their apparent diversity and
universality. When viewed from the outside, low-power laser therapy seems to be a panacea. If one
considers that low-power laser effects are attributable to light action on the respiratory chain, their
diversity as well as their universality can be explained quite easily.
The respiratory chains for complex eukaryotic (Figure 7.3a) and prokaryotic cells differ markedly.
The carriers can have different structures, and, in the case of prokaryotic cells, the respiratory chains
usually branch with alternative electron transfer pathways to multiple terminal acceptors. There are
also some minor differences in respiratory chains of complex (e.g., mammalian) cells and primitive
eukaryotic cells (yeasts). Despite these differences, the functional principles of the chains as well as
the operation of carriers are universal. Precisely this fact makes low-power laser effects so diverse, at
least on a cellular level.
An irradiation-induced alteration in the overall cellular redox state is considered a key step in secondary
reactions (Section 7.2.3), but it does not explain all irradiation effects at a first glance. It is well docu-
mented that more than one function of cells can be influenced by irradiation. For example, some authors
have described an increase in the proliferation of fibroblasts (Boulton and Marshall, 1986; van Breugel
and Dop Bär, 1992; Nara et al., 1992; Loevschall and Arenholt-Bindslev, 1994b) and keratinocytes
(Steinlecher and Dyson, 1993; Loevschall and Arenholt-Bindslev, 1996). Others have found that irradi-
ation increased the motility of fibroblasts (Noble et al., 1992) and keratinocytes (Haas et al., 1990),
collagen synthesis in fibroblasts (Lam et al., 1986; Balboni et al., 1986), taxis of fibroblasts (Albrecht-
Büheler, 1991) and autocrine production of bFGF (a growth factor) by fibroblasts (Yu et al, 1994). Analysis
of this data clearly showed that when one cellular function was altered by irradiation, the others remained
unchanged. For example, when the motility of keratinocytes changed, no effects on their proliferation
were noticed (Haas et al., 1990). When the proliferation of keratinocytes was increased, no significant
effects on their migration were found (Loevschall and Arenholt-Bindslev, 1996). The fibroblasts that
responded to light by increasing collagen synthesis did not increase proliferation (Lam et al., 1986; Balboni
et al, 1986). In fact, the production of collagen type I by fibroblasts was affected by irradiation in an
inverse manner to cell proliferation; when cell proliferation increased, collagen type I production
decreased (van Breugel and Dop Bär, 1992). It should be noted that collagen synthesis by irradiated
fibroblasts was found to be ascorbate-dependent, i.e., connected to redox events (Labbe et al., 1990).
Another fact in connection with this: In experiments with fibroblasts, procollagen production was, on
average, enhanced approximately fourfold by He-Ne laser radiation. The greatest enhancement (36-fold)
was noted in cultures that initially synthesized procollagen at a relatively low level, while an insignificant
effect was achieved in cultures that already actively synthesized the procollagen (Lam et al., 1986).
Explaining such a diversity of possible responses of one cell type to monochromatic light is not trivial.
A possible explanation in the case of a prokaryotic E. coli cell could be as follows. Stimulation of E. coli
growth by monochromatic visible light has been suggested to be a ∆pH-dependent genetic process
(Tiphlova and Karu, 1991a). However, growth stimulation is not the only process utilizing the pH
gradient. A priority in the utilization of ∆pH for ATP synthesis, nutrient transport, taxis, and initiation
of genetic processes has been proposed. This priority enables the metabolic processes utilizing ∆pH to
be regulated and coordinated (Tiphlova and Karu, 1991a).
How the priority between motility, secretory and replicative functions in fibroblasts or keratinocytes
is regulated to enable a cell to respond in a certain way is not yet understood. Solving this problem will
have a great impact on the understanding of low-power laser effects.
7.3.3 Biological Limitations of Low-Power Laser Effects

It is well known that cultured cells are heterogeneous with regard to proliferative activity of subpopula-
tions (Nias, 1968; Azzarone and MacLeira-Coelho, 1982; Hassel and Stanek, 1983). There is no reason
to believe that all subpopulations and even all individual cells respond to irradiation in an absolutely
similar way. The following examples illustrate this suggestion.
One conclusion from experiments with cultured cells was that only the proliferation of slowly growing
subpopulations can be stimulated by irradiation (Karu et al., 1987b). Also, the experiments with HeLa
cells demonstrated that one of the effects of He-Ne laser irradiation of these cells is a decrease of the
duration of the G1 period (Karu et al., 1987b). It means that the cells in the G1 phase of the cellular cycle
certainly responded to irradiation in these experimental conditions.
The results of experiments with E. coli grown in various growth media showed that all E. coli batches
contained a subpopulation that, in response to irradiation, rapidly began a new cycle of replication and
division (Figure 7.14). The number of cells in this subpopulation depended on the cultivation condi-
tions, being smaller in faster-growing populations (e.g., the glucose-grown culture), and larger in slower-
growing populations (e.g., the arabinose-grown culture). First, one can suppose that, in cells of this
light sensitive subpopulation, the particular metabolic state necessary for the division could be estab-
lished for the same reason, and second, that the irradiation helped them to achieve this active state.
Also, this set of experiments (Tiphlova and Karu, 1991b) clearly proved that there is a limit in the
specific growth rate of all populations (0.80 h-1) that is not dependent on growth conditions, and it is
not possible to stimulate the populations that are already growing at this rate. This was also found to
be the reason E. coli growth was not stimulated in summer but the effect was maximal in winter. In
autumn and winter, the intact culture featured a relatively slow growth. In spring and summer, when
the growth of that culture accelerates and the growth rate of the control culture is almost comparable
to that of the culture exposed to the optimum dose of red light in the autumn-winter period, the
irradiation had but little effect (Karu, 1989a).
In principle, a similar effect of a biological limit was found when the luminol-amplified chemilumi-
nescence was measured in murine spleen cells after He-Ne laser irradiation and treatment with an object
of phagocytosis Candida albicans (Karu et al., 1989). The irradiation effect was detectable in these cases
only when the cells were treated first with a low concentration of Candida albicans (5×107 particles/ml
in our experimental conditions). At the concentration of 1 × 108 particles/ml the chemiluminescence was
maximally activated, and no additional activation by light was possible.
Electron microscopy studies of human lymphocytes demonstrated that, after He-Ne laser irradiation,
changes in the structures of nuclei existed in ≈ 70% of the cells (Manteifel and Karu, 1992). Only 25–47%
of 3T3 fibroblasts responded to near IR radiation by extending their pseudopodia toward the monochro-
matic light source. Importantly, in these experiments, the percentage of reactive cells depended on the
irradiation wavelength (Albrecht-Brüeheler, 1991).
Some cell types were found to not respond to the irradiation. For example, the silent neurons of Helix
pomatia did not respond to He-Ne laser irradiation, while the spontaneously active neurons had a strong
response under the same experimental conditions (Balaban et al., 1992).
In some cases, it is possible to cause only a partial activation of cells. An example here is the mitogenic
activation of human peripheral lymphocytes. A summary of these experiments is presented in Figure 7.15.
The irradiation of lymphocytes with a He-Ne laser (56 J/m2, 5.6 W/m2, 10 s) induced short-term changes
in these cells, both qualitatively and quantitatively similar to those caused by the mitogen phytohemag-
glutinin (PHA). There was a two- to threefold increase in intracellular Ca2+ concentration 2 min after
either irradiation or PHA treatment. Enhanced acridine orange binding to chromatin (reflecting the
changes in the structure of chromatin) and increased RNA synthesis were both recorded, with maximal
FIGURE 7.15 Comparison of lymphocyte activation by phytohemagglutinin and He-Ne laser radiation (Fedoseyeva
et al., 1988b, Karu et al., 1991b; Manteifel and Karu, 1992; Shliakhova et al., 1996).
values occurring 1.5–2 h after the treatment. These findings indicate an enhanced template activity of
chromatin. Ultrastructural changes in the nucleoli of both irradiated and PHA-treated cells evidenced
an intensification of rRNA metabolism, including its synthesis, processing and transport. It was concluded
that there is a similarity in the transcription activation of r-genes under PHA action and He-He laser
irradiation. Both the irradiation and PHA treatment caused an accumulation of preliminary terminated
proto-oncogene c-myc RNA in the lymphocytes, but caused no variation in the amount of full-length
c-myc RNA. Despite similarities in the early cell response to PHA and irradiation, the irradiated lym-
phocytes did not enter the S-phase, i.e., no full mitogenic activation occurred in the irradiated lympho-
cytes. In contrast to the PHA that is continuously present in the cellular suspension throughout mitogenic
activation, the laser light is on for only 10 s. This period is apparently not long enough for the light
stimulus to cause the entire cascade of reactions needed for blast transformation. Detailed data about
lymphocyte activation can found in the papers of Karu (1992b, 1996b).
Direct measurement of ionic currents through a cell membrane under He-Ne laser radiation using
the patch-clamp technique showed that no light effects on the voltage-activated whole-cell ionic currents
were found in any type of the cells studied (rat spinal cord neurons, rat hippocampus pyramidal neurons,
guinea pig cardiomyocytes, rat glial cells, bovine pulmonary artery endothelial cells). The only ionic
current type influenced by the radiation in all the cells studied was the background single-channel current
recorded in the cell-attached confirmation of the patch pipette (Karu et al., 1996b).
A greater quantity of nitric oxide (NO) was produced by mouse peritoneal macrophages after 660 nm
irradiation only in the cases where the cells were cultivated in the presence of a special NO inducer
medium. Irradiation without the NO inducer medium had no effect on enhancing NO production (Naim
et al., 1996).
Only partially inactivated Na+, K+-ATPase was reactivated by irradiation with a 904 nm diode laser,
while, at the same time, laser irradiation did not stimulate the activity of the native enzyme (Bolognani
and Volpi, 1991). The photoreactivation of superoxide dismutase and catalase by radiation λ = 632.8 nm
was significant only in conditions where the pH of these solutions was reduced before irradiation
(Zubkova, 1978; Gorbatenkova et al., 1988). These examples clearly prove that it is not possible to activate
a process that is activated already or occurring at speed near maximal. This conclusion underlines, once
more, that laser biostimulation is not a general phenomenon, but occurs only in certain circumstances.
Bertoloni et al. (1993) established that not all strains of E. coli displayed an appreciable photoresponse
to He-Ne laser irradiation. Only those cells whose normal rate of growth was particularly slow due to
the presence of factors inhibiting cell reproduction were stimulated. Table 7.2 presents the E. coli strains
studied by Bertoloni et al. (1993). The strains whose growth was stimulated by He-Ne laser radiation are
also marked in Table 7.2.
It is quite possible that some genetic factors are involved in the photosensitivity of E. coli strains. This
suggestion is supported by data from Voskanyan et al. (1985, 1986). They found that the radio-protective
action of He-Ne laser radiation was genotype-dependent in the case of E. coli K-12 mutants AB1157 and
Gam 444. Also, a fast-growing wild strain of E. coli was not stimulated by He-Ne laser radiation (G.Ber-
toloni and G.Jori, personal communication).
TABLE 7.2 E. coli Strains Derived from K12 in which Photosensitivity was Investigated (Modified from
Bertoloni et al., 1993)
Growth Stimulation
Log-Phase Plateau Phase
No. Strains Genotypes Culture Culture
1. db1344 araD, argF-lac, flb, pts, relA, rpsL, lamB(am), deoC + +

2. 921 thr, leu, met, las, nal, tonA, hsdR + –
3. J53 pro, met + –
4. AB1157 thr, leu, pro, arg, his, thi, lac, gal, rpsl, supE + –
5. db1229 his, trp, las, Sm, Tn10 + +
6. db1245 Sm, ura, arg, thi, his, ade, lac – –
7. CGSC6405 araD, argF-lac, flb, non-9, gyrA, relA, resL, metE, btu: Tn10, + –
thi, deoC
In the case of yeasts, not all strains were stimulated equally. It was found that the degree of protein
synthesis stimulation was in agreement withthe liability of metabolism, i.e., with the possibility of
reconstructing the metabolism (Fedoseyeva et al., 1988a). Also, not all mammalian cell lines cultivated
in vitro responded to irradiation in equal measure; some did not respond at all (Marchesini et al., 1989).
All this data indicates, again, that laser biostimulation is not a general phenomenon, and serious
biological limits exist. Recall also that the effects depend on light parameters (Karu 1989a, 1998), and
last, but not least, biochemical and morphological changes in the irradiated cells depend on the time
elapsed after the irradiation. This means that the measurements should be made at the right time. For
example, a significant increase in DNA synthesis of E. coli WP2 trp– was detectable only during the first
10 min after inoculation (Tiphlova and Karu, 1991a) and growth stimulation was maximal 1 h after the
irradiation and vanished later on (Tiphlova and Karu, 1988). When irradiating the yeasts, the irradiation
effect was revealed in the exponential phase of culture growth (Fedoseyeva et al., 1984). At the same
time, the growth stimulation effect of E. coli WP2 was detectable only in the lag phase of growth (Tiphlova
and Karu, 1988,1991a,b). All these limits, limitations and difficulties should be taken into account when
planning an experiment.
7.3.4 Dependence on the Physiological Status of the Host Organism

Such a complex substance as human blood reacts to irradiation as a dependence on the physiological
(immunological) status of the host organism. The measurements performed on whole blood of 28
clinically healthy people, showed that laser irradiation (820 nm, 292 Hz, 1×104 J/m2) had no statistically
significant effect (stimulation or inhibition) on the luminol-amplified chemiluminescence (CL) of healthy
human blood (Karu et al., 1995b).
The situation was quite different when the CL of the blood from a healthy female donor was recorded
during periods of acute viral respiratory illness (Karu et al., 1993b). Note that CL reflects the intensity
of free radical reactions (Baggiolini and Wyman, 1990).
On the organism level, where complex regulation mechanisms are involved and more than one cell
type is irradiated simultaneously, the situation is more complicated. In some cases, the effects of irradi-
ation can be not only stronger or weaker, as discussed above, but can even change its nature (i.e., being
alternately suppressive or stimulative for radiation with constant parameters). In the example given below,
the irradiation effect (both its nature and magnitude) is correlated with percentage variation of cellular
components in a complex mixture (Karu et al., 1993a).
A murine spleen suspension consists mostly of lymphocytes (∼60–85%) and neutrophils (∼10–15%).
Other cell types (monocytes, eosinophils, basophils, plasmacytes, etc.) are generally present at ∼0.1–2%.
These cellular percentages vary during illnesses and with the age of the organism (Cheville, 1983).
Spleen suspension from A/Sn mice of two different age groups (1.5–2 months and 8–9 months old)
was irradiated with a laser diode at 820 nm (292 Hz, 1.1 × 103 J/m2) and luminol-amplified CL was
recorded. It was possible to find correlations between the cellular composition in the spleen suspension
and the irradiation effect. The linear regression analysis revealed direct correlations between the
irradiation effect and the percentage of plasmacytes (p < 0.001), neutrophils (p < 0.001), myelocytes
(p < 0.01), and an inverse correlation between the irradiation effect and the percentage of lymphocytes
(p < 0.001) (Figure 7.16).
Thus, an increase in the percentage of neutrophils, neutrophil precursors, and plasmacytes, as well as
a decrease in the percentage of lymphocytes, was found to increase the irradiation effect (increased CL).
It is known that plasmacytes develop from activation and transformation of B lymphocytes on contact
with antigens. The amount of neutrophils and plasmacytes in spleen is known to increase with aging
and as a result of chronic infections, as well as other diseases (Cheville, 1983).
As one can see from the correlations shown in Figure 7.16, there are regions (i.e., respective cellular
compositions of the spleen) of CL stimulation and inhibition as well as a rather broad range of cellular
compositions between the two. One should also note that, using the correlation equations shown in
Figure 7.16, it is possible to predict the effect of irradiation (820 nm, 292 Hz) when the cellular compo-
sition is known. On the other hand, it is also possible to make some predictions about cellular composition
when the irradiation effect is known.
7.4 Clinical Applications of Low-Power Laser Effects
7.4.1 Focus of Application

Low-power laser therapy is now used by physiotherapists (treatment of a wide variety of acute and
chronic musculoskeletal aches and pains), dentists (treatment of inflamed oral tissue, healing of diverse
ulcerations), dermatologists (treatment of edema, indolent ulcers, burns, dermatitises), rheumatologists
(attenuation of pain, treatment of chronic inflammations and autoimmune diseases), and by other
specalists as well as general practitioners. This modality is also used rather widely in veterinary medicine
EFFECT OF IRRADIATION %
220
160
100
r=0.65 p<0.001
y=7x+61
40
0 6 12 18 24
NEUTROPHILS, %
220
160
100
r=0.74 p<0.001
y=34x+72
40
0 1 2 3 4 5
PLASMACYTES, %
220
160
100 r=0.51 p<0.01

y=5x+84
40
0 10 20 30
MYELOCYTES, %
220
160
100
r=-0.59 p<0.001
y=2.6x+326
40
40 60 80 100
LYMPHOCYTES, %
FIGURE 7.16 Linear regression analysis between percentage of (A) neutrophils, (B) plasmacytes, (C) myelocytes, or
(D) lymphocytes in cellular suspension and the effect of the irradiation (820 nm, 292 Hz, 1×103 J/m2) r denotes the
coefficient of the correlation (after Karu et al., 1993a).
(especially in racehorse training centers) and in sports medicine and rehabilitation clinics (reduction
of swelling and hematoma, relief of pain and improvement of mobility, treatment of acute soft tissue
injuries). For more details, the following books are recommended: Baxter (1994); Oshiro and Calderhead
(1988,1991); Calderhead et al. (1991); Triner et al. (1999). Some areas where the most experimental
and clinical work has been done are in wound healing, injured nerve regeneration, pain attenuation
and treatment of diverse rheumatological conditions.
The clinical effects of light can be classified as either direct or indirect, depending on whether
the light causes an effect to occur within the irradiated tissue or whether a nervous or neuroen-
docrine signal is generated in the irradiated area and causes a systemic effect in another part of
the body.
As a whole, the field of low-power laser effects is diverse. The versatility of this kind of treatment has
been a controversial and poorly explained feature, making “laser biostimulation” an uncertain medical
modality. Some possible explanations for the controversies of low-power laser effects were presented in
Section 7.3.
7.4.2 Is Low-Power Laser Therapy a Mature Modality?

The clinical field of low-power laser therapy is characterized by a variety of diverse methodologies, and
often by a lack of double-blind tests or even control groups. Moreover, the laser exposure parameters
used (dose, wavelength, intensity, CW or pulsed mode, pulse duration and repetition rate) vary widely.
In many cases, it has not been proven that laser therapy is better than alternative therapeutic treatments.
Moreover, the operative mechanism on an organism level has not been identified.
The conclusion from the preceding is that low-power laser therapy is not yet a mature modality. It is
still developing.
7.4.3 Possible Optimal Light Parameters for Low-Power Laser Therapy

In the early days of low-power laser therapy, He-Ne lasers in the 1–30 mW power range were used
exclusively. These are still employed today, although currently, a large range of additional equipment
varying from single diode probes to multiple diode heads and multihead systems is manufactured and
marketed as well.
Such intense activity by the commercial manufacturers is surprising for two reasons. First, low-
power laser therapy, not yet a mature medical modality, is out of the mainstream of medical applica-
tions. Second, the optimal parameters of light needed (wavelength, intensity, CW or pulsed mode,
pulse width, repetition rate, and duty cycle) are not clear, and quite probably vary for different medical
applications.
On the laser therapy equipment market, one can now find semiconductor lasers operating at a wide
variety of wavelengths (660–1300 nm), pulse frequencies (2–30,000 Hz), and output powers (1–100 mW).
The combined action of various wavelengths is used in numerous diode heads and other integrated
equipment (e.g., an He-Ne laser–IR diode laser combination). Multi-wavelength effects have been very
poorly investigated to date, and the possible benefit of such equipment is not clear. In fact, one should
be careful in using simultaneous irradiation at certain wavelengths. For example, it has been found that
the simultaneous dichromatic irradiation of a cellular monolayer with light in the red and far red regions
produced a substantially reduced or completely absent response (Figure 7.4) despite the fact that each
wavelength-stimulated proliferation when used alone (Karu et al., 1984a,b).
Much experimental work should be done before optimal parameters for clinical applications can be
recommended. Discussed below are some considerations stemming from experiments at the cellular level.
An ideal laser for low-power therapy must emit a beam at a wavelength capable of reaching not only
superficial tissue but also deeper layers. In addition, this wavelength must provide a photoexcitation of
photoacceptor molecules to give a maximal photobiological response. These two objectives can be met
by analyzing the action spectra of various photobiological effects, absorption spectra of both photoac-
ceptor molecules responsible for these photobiological effects and the tissue chromophores, and wave-
length penetration depth data. As no action spectra for clinical effects have yet been produced, action
spectra for cellular effects must be used. Figure 7.17a presents three of these, and Figure 7.17b illustrates
the absorption spectrum of a possible photoacceptor for low-power laser effects. Figure 7.17c presents
absorption spectra of the main tissue chromophores in red–far red regions, and Figure 7.17d depicts skin
penetration depth data for light at these wavelengths.
Analysis of the data presented in Figure 7.17 indicates that the optimal wavelengths are near 760 nm
and between 810–840 nm. In both regions, the tissue chromophores have a weak absorption
(Figure 7.17c) and light penetration into the skin is near maximal (Figure 7.17d). To illustrate the deep
penetration of light into tissues, recall that ~2% of incident light at 580–600 nm reaches the uterine
lumen in pregnant rats, and this is believed to be enough to influence physiological processes related to
circadian rhythms (Jacques et al., 1987).
The cytochrome c oxidase (cyt a/a3) has an absorption maximum in the oxidized form at 830 nm
(extinction coefficient = 1.4 mM cm-1) due to the copper components of this molecule (Wharton and
Tzagaloff, 1964). Three points should be emphasized here:
1. The absorption of cyt a/a3 near 830 nm is weak, but sufficient to cause photochemical reactions.
In some tissues (e.g., myocardium, brain), the density of mitochondria is high, which also results
in a higher cyt a/a3 concentration. This circumstance enables redox states of the mitochondria to
be measured in situ using a fiberoptic based spectrophotometric technique (Rea et al., 1985).
2. Figure 7.17b presents the absorption spectrum of cyt a/a3 (cytochrome c oxidase) in the oxidized
form insofar as the absorption spectra of its redox intermediates are not yet available. Recall that
some absorption spectra of a monolayer of HeLa cells were presented in Figures 7.6 and 7.7, as
well as in Karu et al. (2001). It was supposed that these spectra belong to a redox intermediate of
cytochrome c oxidase. Recall also that the fully oxidized form of cytochrome c oxidase appeared
to be insensitive to He-Ne laser radiation (Pastore et al. 2000).
3. Recall that, to be light sensitive, the electron flow and ATP synthesis must be coupled (Kato et al.,
1981). Irradiation experiments should be performed with cells or mitochondria with functioning
respiratory chain. It could be expected that the irradiation of cristallic cytochrome c oxidase (or
its solution), which became available recently (Gennis and Ferguson-Miller, 1995), is not a useful
experiment to explain the mechanism of low-power laser therapy.
FIGURE 7.17 An evaluation of potentially optimal wavelengths for low-power laser therapy: (a) action spectra for
three photobiological effects — (DNA and RNA synthesis rate in plateau-phase HeLa cells (Karu et al., 1984b) —
and chemiluminescence (CL) emittance by murine splenocytes (Karu et al., 1993b); (b) absorption spectra of
suggested photoacceptor in eukaryotic cells, cytochrome c oxidase (Wharton and Tzagaloff, 1964), (c) absorption of
principal tissue chromophores; (d) penetration depth of various light wavelengths into tissue (Sliney and Wolbarsht,
1980).
Taking all this into consideration, laser devices emitting at 820–830 nm seem to have wavelengths
sufficient for low-power therapy. Lasers operating at another promising wavelength, 760 nm, only recently
became commercially available. Also, 680 nm wavelength is good, with the additional benefit for clinicians
that this wavelength is visible by eye, which facilitates the irradiation procedure. Quite possibly, for
different applications, different wavelengths are optimal.
In experiments with mammalian cellular monolayers, optimal doses in the red and far red regions
were found to be near 102 J/m2 (Karu et al., 1984a,b). During irradiation of real tissues, the doses are
necessarily higher due to light losses through reflection and scattering. For example, it has been suggested
that, in the case of peptic ulcer treatment, the clinical dose providing an equivalent response to the
laboratory 102 J/m2 effect should be approximately 400 times higher (Karu, 1989a).
Experiments with cellular cultures, E. coli and identified neurons have shown that, in many cases, the
intensity of light is a more important parameter than the total dose (reviews of Karu, 1989a, 1998). In
other words, these experiments demonstrated that one of the rules of photochemistry, the reciprocity
rule (Bunsen-Roscoe law), is often not valid. The reciprocity rule says that an effect does not depend on
the intensity or the irradiation time when the dose is constant. This issue of the importance of the light
intensity was specifically investigated and it was found that at least two reaction channels (mechanisms)
are involved in producing the same photobiological macroeffect (Karu et al., 1994b). One channel was
activated by a certain dose, and the reciprocity law was valid in that case. A second channel was activated
by a certain irradiation time (independent of dose), and therefore, the reciprocity rule was not valid in
that case.
In experiments where the reciprocity rule was not valid, a strict threshold (e.g., near 6 W/m2 at 633
nm (Karu et al., 1984a) and a very narrow range of optimal intensities was recorded in the light intensity
vs. cellular response relationship. The issue of optimal intensities at different wavelengths for various
medical applications has not been addressed. On an empirical basis, it has been proposed that, for CW
830 nm diode lasers, the ideal power output is ~60 mW (Moore and Calderhead, 1991).
Other unanswered questions include whether medical devices should employ continuous or pulsed
laser radiation, and, if pulsed radiation has some benefit, what are the optimal pulse frequencies, durations,
and duty factors? Examples presented by Karu (1998) for various cellular systems clearly demonstrate the
importance of laser parameters associated with pulsed operation. However, it is not yet clear that a pulsed
source has a specific clinical benefit over a CW device producing the same wavelength and average power
density. Quite probably, the optimal laser light parameters associated with pulsed operation are different
for different clinical applications.
7.5 Summary
Biological responses of cells to visible and near-lR (laser) radiation occur due to physical or chemical
changes in photoacceptor molecules, components of respiratory chains like NADH-dehydrogenases and
cytochrome c oxidase. As a result of the photoexcitation of electronic states, the following physical or
chemical changes can occur:
• Alteration of redox properties and acceleration of electron transfer
• No release from the catalytic center of cytochrome c oxidase
• Changes in biochemical activity due to local transient heating of chromophores
• One-electron auto-oxidation and O2•− production
• Photodynamic action and 1O2 production
The primary physical or chemical changes induced by light in photoacceptor molecules are followed
by a cascade of biochemical reactions in the cell that need no further light activation and occur in the
dark (photosignal transduction and amplification chains). These reactions are connected with changes
in cellular homeostasis parameters. The crucial step here is thought to be an alteration of the cellular
redox state — a shift toward oxidation is associated with stimulation of cellular vitality, and a shift toward
reduction is linked to inhibition.
Cells with a lower than normal pH, where the redox state is shifted in the reduced direction, are
considered to be more sensitive to the stimulative action of light than those with the respective parameters
optimal or near optimal. This circumstance explains the possible variations in observed magnitudes of
low-power laser effects. Light action on the redox state of a cell via the respiratory chain also explains
the diversity of low-power laser effects. Beside explaining many controversies in the field of low-power
laser effects (i.e., the diversity of effects, the variable magnitude or absence of effects in certain studies),
the proposed redox-regulation mechanism may be a fundamental explanation for some clinical effects
of irradiation, for example, the positive results achieved in treating indolent wounds, chronic inflamma-
tion, and ischemia, all characterized by acidosis and hypoxia.
One unsolved mystery is integral to low-power laser effects. It is rather well documented that more
than one of the cellular functions (e.g., the replicative, motile, and secretory functions of fibroblasts or
keratinocytes) can be influenced by radiation at the same wavelength. Even more interesting is that, when
one of these cellular functions is altered by irradiation, the others remain unchanged (Karu, 1998). This
finding clearly indicates the existence of some unknown regulatory mechanism establishing priorities in
a cell. Identifying this priority regulation system (i.e., the preferential utilization of light-generated proton
motive force, ATP, etc.) will have profound importance for future studies of low-power laser effects.
Finally, a remark about the reliability of low-power laser effects should be made. Years ago, a rather
usual perception existed that laser biostimulation occurred in Eastern but not Western laboratories. At
that time, the actual situation was indeed rather close to this. Very few Western scientists took laser
biostimulation seriously, and even fewer performed experiments. However, lately there have been changes
in this attitude, and a number of well-designed experiments have been performed on various cells. These
data are summarized by Karu (1998).
In some cases, the effects of visible and near IR light on various cellular functions have been described
in papers not dealing with the issue of laser biostimulation at all (e.g., Kato et al., 1981; Chopp et al.,
1990; Albrecht-Büehler, 1991). This circumstance gives a higher reliability to those results for the field
of low-power laser effects.
Alternatively, these findings clearly indicate that the responsivness of mammalian cells to various
wavelengths of monochromatic visible and near IR radiation can be used to understand some physio-
logical processes (e.g., adaptation). Laser biostimulation is only one field where the cellular sensitivity
(which seems to be increased in injured or otherwise stressed systems) to monochromatic visible and
near IR radiation is used. Albrecht-Büehler (1991) described the phenomenon of fibroblast phototaxis
(pseudopodia moving toward a monochromatic light source). In the paper of Kato et al., (1981) it was
suggested that mitochondria in a special part of bird brain could work as photoreceptors for a photobi-
ological process relating to gonadal growth.
To complete this chapter, two remarks from a classical paper by K. Smith (1980) should be remembered.
First, just because humans can’t see through the human body does not mean that the human body is
opaque to all wavelengths of light. Second, because light (especially red light) can penetrate deep into
human tissues, and because absorbed light can cause photochemistry, it is appropriate to be concerned
with the biological consequence of such absorbed light in the tissues of man.
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8
Therapeutic and
Diagnostic Application
of Lasers in
Ophthalmology
8.1 Introduction ....................................................................... 211

8.2 Basic Ocular Anatomy and Physiology............................. 212
8.3 Transmission and Absorptive Properties
of Ocular Tissues................................................................ 214
8.4 Photothermal Laser Applications...................................... 217
Mechanisms of Photothermal Laser–Tissue interaction •
Photothermal Laser Applications • Beam Delivery Systems
for Photocoagulation
8.5 Photodisruptive Laser Applications .................................. 231
Photodisruptive Laser–Tissue Interaction • Photodisruptor
Applications • Photodisruptor Delivery Systems
8.6 Photochemical Laser Applications: Photoablation
and Photodynamic Therapy .............................................. 233
Photoablation Mechanism • Delivery Systems for Photoablative
Lasers • Photodynamic Laser Applications
8.7 Diagnostic Laser Applications ........................................... 239
Visual Acuity Measurement • Laser Doppler Velocimeter •
Keith P. Thompson, M.D. Scanning Laser Ophthalmoscope • Spectroscopic Diagnosis
of Ocular Diseases
Qiushi S. Ren, Ph.D.
8.8 Summary............................................................................. 240
Jean-Marie Parel, Ing., Acknowledgments......................................................................... 241
ETS-G References ...................................................................................... 241
8.1 Introduction
The most widespread medical application for laser technology in medicine has been in ophthalmology.
Since the introduction of the ruby laser more than three decades ago, ophthalmic laser applications have
experienced rapid growth with use of the argon, krypton, argon pumped dye, Nd:YAG, and, most recently,
diode lasers. Lasers have achieved remarkable clinical successes in the treatment of retinal diseases,
glaucoma, and lens capsule opacification following cataract surgery. The 1990s saw another period of
rapid growth of ophthalmic laser technology with the then newly devised excimer and midinfrared lasers.
The reasons for the prevalence of laser applications in ophthalmology are easy to grasp. The eye is one
of the most accessible human organs, and its media (cornea, aqueous humor, lens, and vitreous) are
0-8493-1146-2/02/$0.00+$1.50
transparent to visible light, allowing direct inspection of its internal structures for diagnosis and treat-
ment. Many intraocular tissues contain pigments (such as melanin) that allow absorption of laser energy
for photothermal laser–tissue interactions. Transparent intraocular structures (such as the posterior lens
capsule and vitreous), have also been amenable to laser treatment with Nd:YAG photodisruptors.
Another factor that has facilitated the growth of ophthalmic laser applications is the ability of oph-
thalmologists to evaluate objectively the efficacy of new treatment modalities such as laser therapy.
Whereas efficacy in other medical specialties is more difficult to assess (evaluating new treatments for
patients with cancer or cardiovascular disease requires many patients and years of follow-up), ophthal-
mologists can observe disease processes directly and functional parameters such as visual acuity, visual
field, and intraocular pressure can be measured accurately and objectively. These factors have facilitated
the introduction of new laser treatments for eye diseases and the rapid determination of their efficacy.
This chapter will review the current status of clinical and experimental applications of laser technology
in ophthalmology. Because a basic understanding of ocular anatomy, physiology, and some disease
processes is necessary to understand the rationale for laser applications, a brief overview of these subjects
for the non-physician readership is provided. Also discussed are therapeutic applications of lasers accord-
ing to photothermal, photodisruptive, and photochemical mechanisms of laser–tissue interaction. Cur-
rent diagnostic applications of lasers in ophthalmology are also reviewed.
8.2 Basic Ocular Anatomy and Physiology

The eye is a slightly ovoid organ measuring about 24 mm in length and 23mm in diameter (Figure 8.1).1
It is composed of three distinct tunics or layers. The sclera is the tough outer coat of the eye that makes
up the posterior four fifths of the globe. The sclera is white, because its dense collagen fibrils have variable
diameters that cause diffuse scattering of visible light. Anteriorly, the sclera is contiguous with the cornea,
the clear watchglass-like covering where the collagen fibrils have more uniform diameters and are
arranged in orderly layers, permitting transparency (T ≥ 95% for 400-900 nm; refractive index, n ≈ 1.3765
Figure 8.1 Anatomical drawing of the eye.

Therapeutic and Diagnostic Application of Lasers in Ophthalmology 213
± 0.0005, Tensile strength, T ≈ 106 Nm-2). The middle layer of the eye, composed of a rich network of
blood vessels that supplies nutrition to the eye is termed the uveal tract. In the posterior segment of the
eye, the uvea is termed the choroid. In age-related macular degeneration, the boundary between the
retina and choroid may break down and allow abnormal blood vessels to grow beneath the retina, causing
hemorrhage and loss of vision.2 Anteriorly, the uveal tract is continuous with the iris, the colored aperture
of the eye, which allows light to pass through its central opening (pupil). The diameter of the pupil varies
between 1.5 mm to 8 mm, depending upon the intensity of light reaching the retina.
The eye is divided into three cavities by intraocular structures. The anterior chamber lies between
the cornea and iris. It is filled with a clear water-like fluid, the aqueous humor (viscosity ≈ 1.0 cs; n
≈ 1.3335). Behind the iris and anterior to the lens lies the posterior chamber. The lens of the eye,
composed of specialized crystalline proteins, is clear at birth (n ≈ 1.40-1.42, T ≈ 104 Nm-2) and often
becomes progressively cloudy or cataractous with age. The lens is approximately 0.15 to 0.25 cm3 in
volume, 3.5 mm in thickness, 8 mm in diameter and enveloped in a tough, thin (10–14 µm), trans-
parent, collagenous capsule (n = 1.396, T ≈ 107 Nm-2). The posterior portion of this capsule is left
intact following removal of the lens substance during modern cataract surgery to help support a plastic
intraocular lens. The large cavity posterior to the lens is filled with a transparent jelly-like substance,
the vitreous humor (n ≈ 1.335, viscosity ≈ 10-100 cs). The vitreous body (5.5 cm3) is often surgically
removed for the treatment of diabetic eye diseases and the cavity then fills with aqueous humor, with
no apparent adverse effects upon the eye.
The innermost layer of the eye is the neurosensory retina, an outgrowth of brain tissue that converts
visible photon energy into electrochemical impulses that are processed (in both a digital and analog
fashion) in the inner retinal layers and relayed to the brain via the optic nerve. The outer portion of the
retina contains the photoreceptors and receives its nutrition from the uveal tract, while the inner retina
contains its own retinal vascular supply. Normally, the retinal arterioles are sealed tightly, and allow no
leakage of blood fluids or proteins into the surrounding retina. In diabetes, and several other vascular
diseases, the blood vessels become abnormal and allow fluid to leak into the macula, the posterior region
of the retina that is responsible for central vision, causing retinal dysfunction and loss of vision. Fragile
new blood vessels may also grow onto the surface of the retina, causing both leakage of fluid and
hemorrhage into the eye.3
The eye is internally pressurized with a normal intraocular pressure of about 13 ±3 mm Hg above
ambient pressure. Internal pressure is created by a clear fluid termed aqueous humor, produced by
the ciliary body, a specialized secretory tissue that is contiguous with the retina posteriorly. The aqueous
humor flows anteriorly though the pupil to drain through the trabecular meshwork, a specialized
filtration structure located in the angle between the cornea and iris. Abnormalities of the pressure
regulating system of the eye may result in abnormally high (≥ 22 mm Hg) intraocular pressure, a
condition termed glaucoma, which damages the delicate nerve fibers in the optic nerve, causing a loss
of vision.
The eye has a focal length of about 16.7 mm in air.4 Ophthalmologists prefer to use optical units of
diopters (l diopter = l/F meters) when measuring the refractive error of the eye or the power of the eye’s
optical elements. These are usually determined according to Gulstrand’s reduced schematic eye
(Figure 8.2a).4,5 For instrument design and computational purposes, opticists use the model described
in Figure 8.2b.6 Two thirds of the eye’s optical power, or about 44 diopters, results from the curvature of
the cornea; the remainder is provided by the crystalline lens. The lens can change shape and increase its
focusing power for near vision (accommodation) until about the fifth decade of life, when, for most
people, reading glasses become a necessity for clear viewing of close objects. Because the cornea is the
predominant optical element of the eye, a small change in its curvature causes a large effect on the total
optical power of the eye, a principle utilized by corneal refractive surgeons.7,8
An emmetropic eye, or one without refractive error, focuses a distant point source of light on the
fovea, the region of the macula with the highest density of cones (1 µm diameter with 1.3 µm spacing),
the photoreceptors that provide the highest resolution (15–30 min. of arc). Nearsighted or myopic eyes
have too much optical power for their length, resulting in displacement of the focal point anterior to the
n=1.33
GULLSTRAND REDUCED F N IMAGE PLAN

SCHEMATIC EYE 16.67mm 5.65mm 16.67mm
22.6mm
Figure 8.2 (a) Optical model of the human eye. (b) Littman-Gullstrand schematic eye with accommodation.
retina. Farsighted or hyperopic eyes have insufficient power for their length, with a focal plane that is
located “behind” the retina.
8.3 Transmission and Absorptive Properties of Ocular Tissues

Light entering the eye can be reflected, scattered, transmitted, or absorbed. Reflected or scattered light
contains information that can be used for noninvasive diagnostic purposes, discussed in Section 8.7. In
ophthalmic laser surgery, the surgeon must choose a proper laser wavelength that is transmitted through
intervening ocular layers to become incident upon the target tissue.
In the visible and near infrared spectrum (400 nm to 1,400 nm), the absorption characteristics of
ocular tissues are determined by a group of chromophores within the tissue. Ocular chromophores
that absorb light in the visible spectrum include melanin, located in the retinal pigment epithelium,
iris pigment epithelium, uvea, and trabecular meshwork; hemoglobin, located in red blood cells
within blood vessels; and xanthophyll, located in the inner and outer plexiform layers of the retina
10,000
n n ton ND:YAG
go go Dye yp
Ar Ar Kr 1064 nm
647.1 nm
577 nm
1
514.5 nm
2
488.0 nm
Extinction coefficient (cm-1)
1000 3
100
4
10
1
400 500 600 700 800 900 1000 1100
Wavelength (nm)
Figure 8.3 Transmission spectra of the major ocular chromophores: melanin (1), reduced hemoglobin (2),
oxygenated hemoglobin (3), and macular xanthophyll (4). Also shown are the wavelengths of four commonly utilized
ophthalmic lasers: argon, krypton, dye and Nd:YAG. (Permission pending.)
in the macular region. The light absorption spectra of these pigments is illustrated in Figure 8.3. In
the mid-infrared spectrum (1.8 µm–10.6 µm), the major molecule absorbing incident photons is
water (Figure 8.4).
The retina contains at least six pigments: melanin, hemoglobin, macular xanthophyll, rhodopsin, cone
photopigments, and lipofuscin. The first three of these are of major importance in laser photocoagulation
of the retina.
The cornea consists of five principal layers, the epithelium, Bowman’s layer, stroma, Descemet’s mem-
brane and the endothelium (Figure 8.5). The cornea is protected continuously by a tear film that has a
high lipid content and a high index of refraction (n = 1.40). Due to transmission characteristics, the
cornea provides an effective window for vision, photocoagulation, photodisruption, imaging and exam-
ination of intraocular structures (Figure 8.6).
The transmission and absorption characteristics of the crystalline lens change with age (Figure 8.7).
The total transmittance at the various anterior surfaces is given in Figure 8.8.
104
absorption coefficient α (cm-1)
H2O
103
102
10
1000 2000 3000 4000 5000 7000 10000 λ (nm)
wavelength
Figure 8.4 Absorption spectra of water.

Figure 8.5 Detailed structure of the cor-

nea.
100
80
1
PERCENT TRANSMITTANCE
60 2
40
CORNEA TRANSMITTANCE
TOTAL
1 DIRECT, 4 1/2 YRS.
20 2 DIRECT, 53 YRS.
300 400 500 600 800 1000 1200 1600 2000

Figure 8.6 Corneal transmittance spectrum. WAVELENGTH MILLIMICRONS
100
1
80
2
TRANSMITTANCE
LENS TRANSMITTANCE
60
TOTAL , 4 1/2 YRS.
1 DIRECT, 4 1/2 YRS.
2 DIRECT, 53 YRS.
3 DIRECT, 75 YRS.
40
3
PERCENT
20
Figure 8.7 Lens transmittance spectrum 300 400 500 600 800 1000 1200 1600 2000
(aging effect). WAVELENGTH MILLIMICRONS
100
80 2
PERFECT TRANSMITTANCE
60
4
40
TOTAL TRANSMITTANCE
AT THE VARIOUS ANTERIOR
20 SURFACES
1 AQUEOUS 3 VITREOUS
2 LENS 4 RETINA
300 400 500 600 800 1000 1200 1600 2000

WAVELENGTH MILLIMICRONS
Figure 8.8 Total transmittance of the entire eye.
8.4 Photothermal Laser Applications
8.4.1 Mechanisms of Photothermal Laser–Tissue interaction

Photothermal laser–tissue interactions occur when laser energy is absorbed by the target tissue and
converted into heat. The effect on the tissue depends on both the magnitude and rate of temperature
elevation. Low (less than 10ºC) temperature elevations that occur over many seconds to minutes
(photoheating) usually cause cell damage or death without causing structural alterations to the
tissue. Greater temperature elevations (20–30ºC), occurring over shorter time intervals (approxi-
mately 1 sec.), cause thermal coagulation of tissue (photocoagulation), with cellular death and
irreversible structural damage to the tissue due to denaturation (loss of the three-dimensional
molecular structure) of tissue proteins.9 Laser energy depositions that rapidly (much less than 1
sec.) heat the tissue above its boiling point cause thermally mediated tissue removal by explosive
vaporization (photovaporization). The wavelength of laser radiation, the absorption coefficient of
the tissue, the power (or energy) density, and the duration of laser radiation determine which of
these photothermal effects predominates.
To minimize unwanted thermal damage to surrounding ocular tissue during laser treatment, a wave-
length should be selected that is preferentially absorbed by the target tissue, and the laser exposure
duration should be shorter than the thermal relaxation time of the tissue, given by:
T h = d2 / 4k
where d represents the absorption depth of the radiation, and k is the thermal diffusivity of the tissue.
For example, mid-infrared (2.94 :m) Er:YAG and Er:YSGG lasers are under investigation for cutting
the cornea. Water (k = 1.5 x 10-3 cm2/sec) is the primary absorbing molecule at this wavelength (d =
1 µm) and the thermal relaxation time is calculated to be 1.7 µs. Therefore, to achieve efficient
photovaporization with minimum damage to the surrounding corneal tissue, the pulse duration of
the laser must be significantly less than 1.7 µs. Decreased thermal tissue damage with shorter laser
pulse duration has been demonstrated by comparing the tissue damage adjacent to corneal excisions
created by a Q-switched (pulse duration = 100 ns) to a free running (pulse duration 250 µs) Er:YSGG
laser (Figures 8.9a and b).
Figure 8.9 (a) Er:YSCG normal mode (250 µs pulses). (b) Er:YSCG Q-switched mode (100 ns pulses). Light
microscopy of corneal excisions produced by a mid infrared Er:YSCG laser demonstrates the effect of pulse duration
on adjacent thermal damage to tissue. Excisions produced in the normal mode (250 µs) causes significantly more
thermal damage than when operated in the Q-switched mode (100 ns).
8.4.2 Photothermal Laser Applications

Tables 8.1a and 8.1b summarize the present clinical and research applications for photothermal lasers in
ophthalmology, respectively.
Table 8.1a Photothermal Laser Applications in Clinical Use

Laser/Wavelength
Procedure Disease (nm) Rationale Comment
Pan-retinal Proliferative Argon-488/514 Destruction of peripheral Widespread
photocoagulation retinopathies Krypton 647 retina decreases stimulus for application
Dye-577 new blood vessel growth
Focal macular Diabetic macular Argon-488/514 Focal treatment decreases Widespread
photocoagulation edema, vein Krypton 647 leakage of fluid in retina application
occlusions Dye-577
Photocoagulation of Age-related Argon Green-514 Destruction of new vessel Widespread
subretinal macular Dye-577 membranes prevents application
neovascular degeneration complication from
membranes subretinal hemorrage and
leaking vessels
Traeculoplasty Open angle Argon-488/514 Focal photocoagulation of Compared
glaucoma trabecular meshwork favorably to
improves aqueous outflow, medications in
decreasing pressure recent study
Peripheral Narrow angle Argon-488/514 Hole in iris allows alternative Has replaced need
iridotomy glaucoma path for aqueous flow, for intraocular
preventing angle closure surgery
Cyclophoto- Advanced Nd:YAG-1064 free Destruction of ciliary body
coagulation glaucoma running thermal decreases intraocular
mode pressure
Table 8.1b Photothermal Laser Applications in Experimental Development

Laser/Wavelength
Procedure Disease (nm) Rationale Comment
Filtration surgery Glaucoma TmHoCr:YAG- Small incision, minimal
2.1µm trauma may increase
survival of filtration bleb
Corneal Corneal scarring HF 2.9 µm Nonmechanical cutting may Full thickness
trephination and disease Er:yAG-2.9 µm decrease postoperative trephination
astigmatism possible in 7 s
Photothermal Hyperopia Ho:YAG Shrinkage of collagen steepens Early clinical
keratoplasty central cornea studies
Laser tissue welding Wound closure Line-tuned HF Fusion of collagen and Unsuccessful in
2.55 µm connective tissue clinical
applications to
date
Cataract removal Cataract HF and other mid- Cataract removal while Will facilitate
IR 2.5–3.0 µm maintaining capsular bag development of
accommodating
lens
Laser-induced Ocular tumors Mid-infrared Selective heating causes tumor
hyperthermia necrosis
8.4.2.1 Photocoagulation Therapies for Retinal Diseases

The use of lasers as a selective heat source to coagulate retinal tissue is one of the most important and
widely adopted ophthalmic laser applications. The 694 nm ruby laser was the first laser introduced to
ophthalmology in the early 1960s.10 Energy from the ruby laser was absorbed by the retinal pigment
epithelium and caused photocoagulation of the outer retina. Unfortunately, this early laser performed
poorly in the clinical setting due to its short pulse duration (≈ 100–300 µsec), its deep penetration depth
(≥ 400µm), variable power output, and crude beam delivery system.
In 1968, the continuous wave argon laser was introduced by L’Esperance.12 This laser emitted at both
488 and 514.5 nm and offered improved beam stability. It quickly surpassed the xenon arc lamp
and ruby laser as the preferred methods for retinal photocoagulation in the 1970s. The irradiance
of the Ar+ laser treatment is approximately 100 W/cm2 with an application time of 0.1–1 second.
Photocoagulation has proven most useful in the treatment of proliferative diabetic retinopathy. In
this disorder, the retina becomes ischemic (starved for oxygen and nutrients) and releases chemical
messengers that are thought to cause the growth of fragile new blood vessels in an attempt to nourish
the oxygen-starved tissue. The abnormal new blood vessels, and their associated proliferating fibrous
tissue, are a major cause of sight-threatening complications in diabetic eye disease. By destroying a
portion of the peripheral retina with the laser, retinal metabolic demands are decreased and the
stimulus for new vessel formation is reduced.3 This treatment, termed panretinal photocoagulation
(Figure 8.10) can significantly decrease the risk for vision-threatening complications from new blood
vessel growth.12–14 The side effects of panretinal photocoagulation — loss of some night vision and
constriction of visual field — are far outweighed by the preservation of central vision offered by
treatment.
The continuous wave Kr+ laser, introduced in 1972, was also used to photocoagulate the retina
effectively, with the additional advantage that the 568 nm wavelength can penetrate ocular media clouded
by cataract or vitreous hemorrhage more efficiently than the 488 nm argon laser.15 In addition, because
568 nm is not absorbed by the xanthophyll pigment located in the macula, the krypton laser is best suited
to treat microaneurysms located close to the fovea.
The widespread use of the argon and krypton lasers by ophthalmologists in the treatment of prolif-
erative diabetic retinopathy has been directly responsible for preserving the vision of many thousands of
diabetic patients.
Figure 8.10 Pan retinal photocoagulation. Multiple 500 µm diameter laser burns have been applied to the periph-
eral retina of this diabetic patient using a 488/514 nm argon laser. Several areas of focal treatment in the macula are
also present.
Laser treatment of the macula was also found to be effective for decreasing the leakage of fluid into
the retina (diabetic macular edema), which also can occur in diabetic eye disease.16 Laser treatment within
the macular region requires special consideration for the absorptive properties of the tissue. Within the
macula, the inner retinal layers contain xanthophyll pigment, which absorbs strongly between 450 and
500 nm. Use of the argon blue wavelength (488 nm) causes heating and destruction of the nerve fiber
layer in the inner retina, resulting in loss of vision (Figure 8.11). By using the argon green wavelength
(514 nm), or a longer wavelength from a krypton or dye laser, absorption by xanthophyll and damage
to the inner retina are minimized.
Figure 8.11 Histology of macula following photocoagulation with the argon blue (488 nm) wavelength. Note the
damage to the inner retinal layers due to absorption of laser energy by xanthophyll pigment. Inner retinal damage
can be avoided by treatment with the argon green (514 nm) or krypton (647 nm) wavelengths.
Treatment of subretinal neovascular membranes that occur in some forms of age-related macular
degeneration is another major application for laser photocoagulators. Macular degeneration is the leading
cause of acquired blindness in the elderly. Here, the surgeon uses a photocoagulator to destroy the
invading vascular membrane, leaving a chorioretinal scar (Figure 8.12). The benefit of photocoagulation
to preserve vision in this disorder has also been demonstrated by a large clinical study.17–21
Tunable dye lasers, introduced to ophthalmology in 1981, gave added flexibility to ophthalmologists
and allowed them to select a wavelength for a given photothermal laser application.22 Ideally, photon
penetration depth for a laser wavelength used in vessel closure should be approximately the same length
as the vessel’s diameter, such that effective bulk heating of the blood column occurs without superficial
damage and perforation of the vessel wall at the radiation site. Treatment of neovascular membranes is
best done with a wavelength maximally absorbed by hemoglobin.
8.4.2.2 Photo-Thermal Laser Treatment for Glaucoma
In addition to treatment of retinal diseases, photothermal lasers have also found widespread clinical
application in the treatment of glaucoma. Continuous wave argon, krypton, or dye lasers are used
to burn a small hole through the iris when narrow angle or angle closure glaucoma is present.
Creating a small opening in the peripheral iris allows an alternate pathway for aqueous humor when
its normal pathway through the pupil is impeded by the lens (angle closure glaucoma). Melanin is
the major absorptive pigment in the iris, and the argon 488 nm wavelength is absorbed quite
effectively (Figure 8.3). In individuals with lightly colored irises, little pigment is present, decreasing
the effectiveness of the laser. In these cases, the use of a Q-switched Nd:YAG photodisruptor,
discussed below, offers the clinician an alternative laser that is independent of pigment absorption
for its effect.23 Creating a hole in the iris with the laser is easily performed with the patient seated
at the slitlamp (Figure 8.13) in the outpatient clinic. This treatment has saved many patients and
surgeons a trip to the operating room and an intraocular surgical procedure with its associated
risks, complications, and costs.
Laser trabeculoplasty is performed in eyes with open-angle-type glaucoma by applying focal photo-
coagulation to the trabecular meshwork located in the angle of the eye.24 For this treatment, the 1–3 W
Figure 8.12 (a) Subretinal neovascular membranes (arrows) in the macula in a patient with age related macular
degeneration. (b) Same eye after photocoagulation treatment with the argon green laser.
argon (488 nm and 514 nm) laser is aimed through a mirrored contact lens and focused into a 50–100
µm spot. Application times of 0.3 to 1 sec. are used. Although the mechanism of action is uncertain, it
is thought that laser photocoagulation may stretch open the structures of the trabecular meshwork,
facilitating outflow of aqueous humor and decreasing intraocular pressure.25 Laser trabeculoplasty may
play a major role in the management of patients with open-angle glaucoma, estimated to be 1.6 million
in the United States alone.26
When medical management fails, glaucoma surgery is indicated to control the intraocular pressure.
The objective of glaucoma surgery is to provide an alternate pathway for fluid to egress from the eye.
The trend in techniques has been toward smaller incisions and less invasive procedures. Recently, a pulsed
TmHoCr:YAG laser (Figure 8.14) has been applied subconjunctivally through a quartz fiber optic probe
for a transcleral filtering procedure.27 In this procedure, a small snip incision is made in the conjunctiva,
approximately 5 mm posterior to the junction of the cornea and sclera (limbus) and the fiber optic laser
probe is passed through the opening and directed over the pupil until the tip approaches the opposite
angle. The beam is shaped and focused by a cylindrical prism built at the end of the probe and directed
toward the limbus to create a full-thickness fistula. The exposure is made with energy ranging from
60–150 mJ per pulse, with pulse duration of 300 ms and a repetition rate of 5 Hz. Total energy levels
required to produce a full-thickness channel ranged from 1.35 to 6.6 J. Compared with conventional
surgical procedures, this technique may avoid the need for large conjunctival flaps and intracorneal
cw : Ar+, Kr+, Nd:YAG,

Ar+ pumped Dye G630
AIGaAs DIODE
and other LASERS
CCD
35mmSLR
SURGEON
PATIENT
BIOMICROSCOPE
FILTERS
Tu-HALOGEN
SLIT-ILLUMINATOR
SLIT APERTURE
Figure 8.13 (a) Typical appearance of a slitlamp laser delivery system. A Keeler diode laser is integrated into the
slitlamp optical system. (b) Optical schematic of slitlamp laser delivery system in widespread clinical use. Laser energy
is conducted to the slitlamp by a fiberoptic cable and directed toward the eye by a coaxial beamsplitter. (continued)
CCD
MACRO 35mm SLR SURGEON
ZOOM
OPTICS
SAFETY
Tu-HALOGEN SHUTTER
50µm FIBER ILLUMINATOR STEREO ZOOM
0.1-0.2 NA OPERATION
MICROSCOPE
STERILIZABLE DM JOY-STICK
PLUG-IN
PREFOCUSSED
CONNECTOR
M2
400µm FIBER
0.2 NA
cw: LASER EYE

Ar+ HeNe ENDOPROBE
Kr+ AIMING
Nd:YAG BEAM
Ar+DyeR630
Al Ga As
&
others FOOT-PEDAL
POWER
LASERS
Figure 8.13 (continued) (c) Typical hand-held operating-room-delivery system.
manipulations, reduce tissue trauma and hemorrhage, and limit the outflow of wound-healing agents
that lead to scarring and failure of the fistula.28 Clinical investigation of this new technique is pending.
Eyes with advanced forms of glaucoma unresponsive to conventional surgical or medical manage-
ment may undergo photocoagulation of the ciliary body (cyclophotocoagulation) with a Nd:YAG
laser operating at 1064 nm in the free running (5–20 ms) mode. Both pulsed (≥10 msec) and
continuous wave Nd:YAG lasers are used to heat and destroy portions of the aqueous humor-secreting
ciliary body in an attempt to decrease intraocular pressure. Diode lasers have also shown promise
for cyclophotocoagulation.29
8.4.2.3 Mid-Infrared Laser Corneal Surgery
Although the healthy cornea is transparent to visible light and does not interact with argon, krypton or
dye photothermal lasers, wavelengths in the mid-infrared and infrared spectrum (1.9 µm–10.6 µm) are
strongly absorbed, largely due to the absorption of water, which composes ≈ 75% of corneal tissue
(Figure 8.15). Within this spectral region, laser parameters such as wavelength, pulse duration, energy
density, etc. can be chosen to cut, shrink, and weld tissue by photothermal mechanisms. Although mid-
infrared corneal laser surgery is still in its infancy, with most techniques still in laboratory or early human
studies, there are many promising potential clinical applications.
8.4.2.3.1 Mid-IR Laser Corneal Cutters (Photocutting)
Corneal and refractive surgery demands a high degree of spatial accuracy and reproducibility. Existing
surgical techniques using metal and diamond scalpels rely heavily on the surgeon’s manual abilities and
experience. Even with the help of high power operating microscopes, manual precision is limited to ≈ 100–200
µm, contributing to the unpredictability of postoperative refractive outcome. Postoperative astigmatism
following corneal transplant surgery continues to be a major problem.30 A mechanical trephine (the
circular blade used by the surgeon to cut corneal tissue for transplantation) produces a torsional moment
on the cornea during cutting, leading to an uncertainty in the precise dimensions of the trephined tissue.
A tissue size disparity between the donor and host of 100 µm produces about 4 diopters of astigmatism.
The application of photothermal cutting lasers to corneal transplant surgery provides the potential
for a noncontact corneal trephination system. Beckman et al.31 demonstrated that the cornea could be
excised with a rapidly pulsed (60–300 Hz) CO2 laser operating at 10.6 µm, emitting a peak power output
of 25,000 watts and an average output power of 150 watts, but the amount of adjacent thermal damage
to the corneal tissue was too excessive for clinical use.31 More recently, Parel, Jeffers et al. selected the
Figure 8.14 (a) Sunrise Technologies’ laser system for glaucoma and (b) its special fiberoptical tip design.
mid-infrared region to investigate laser corneal surgery, and, with a pulsed HF laser (2.7–3.0 µm,
50–200 nsec, 2.4 J/cm2), created 70 µm-wide uniform corneal excisions with much less thermal damage
than that caused by the CO2 laser.37 Therefore, we postulate that the laser might significantly decrease
the amount of undesirable postoperative astigmatism,34 and we have begun comparative studies in
animal models. For clinical application, solid state lasers generating mid-infrared wavelengths are
desirable because of their simplicity, reliability and safety in the operating room. A Q-switched Er:YAG
solid state laser system (2.94 µm, 100 ns, 1.5 J/cm2) coupled to an axicon–lens combination was used
successfully in preliminary corneal trephination studies,35 and research to develop this technology is
continuing.
LIGHT ABSORPTION COEFFICIENT vs. WAVELENGTH

HYDRATED, DEHYDRATED HUMAN CORNEA, and WATER
105
COAG LPTK CUTTING
Absorption Coefficient (1/cm)

104
Co:MgF2
tuning range
103 Er:YAG
(a) (b)
Er:YSGG
102 pulsed HF range
Fully Hydrated Cornea (+-20%)
Dehydrated Cornea (+-20%)
101 Liquid Water (+-10%)
THC:YAG Human Cornea (+-30%)
Nd:YAG
100
1.0 1.5 2.0 2.5 3.0 3.5 4.0
Wavelength (um)
Figure 8.15 Light absorption coefficient vs. wavelength for the hydrated, dehydrated human cornea and water.
Absorption rises sharply for wavelengths longer than 2.5 um with a peak absorption coefficient occurring near 2.9 um.
Because the normal cornea is nearly 75% water by weight, the cornea’s IR absorption spectra closely parallels that of H2O.
Figure 8.16 Circular corneal button excised with pulsed HF laser (2.9 um, 50-200 nsec., 2.4 J/cm2). Edges of the
excised button are smooth and uniform. 360° full thickness trephination is not possible because aqueous fluid
egressing from the first penetration site fills the excision and attenuates energy.
8.4.2.3.2 Laser Photo-Thermal Keratoplasty (LPTK or Photoshrinking)

Because collagen, the major structural component of the cornea, shrinks when heated to 55–58 °C9 the
shape of the cornea and consequently, the refractive power of the eye, may be changed by creating focal
burns in the stroma. Excessive mechanical and thermal damage to the cornea rendered previous attempts
to thermally alter the shape of the cornea by heated metal probes (≥100–600 °C) unsuccessful
(Figure 8.17a). Seiler et al.36–38 recently demonstrated that the refractive power of the cornea can be
adjusted by shrinkage of corneal collagen fibers with lasers emitting around 2 µm.39 A small series of
hyperopic patients treated with the holmium laser coupled to a fiber optic probe delivery system have
shown reasonable predictability and fair stability of the refractive outcome.
By heating the corneal stroma locally with a non-contact mid-infrared TmHoCr:YAG laser system
operating at 2.1 µm in the free-running mode (≈ 300 µsec, 1 Hz), fresh cadaver eyes and the eyes of
rabbits, cats, and non-human primates have been studied topographically and histologically
(Figure 8.17b). The energy density applied to the target varied from 5 to 20 J/cm2 and the total number
of pulses from 5 to 50. Changes of 10 diopters were obtained with five 10 J/cm2 pulses placed in a semi-
Figure 8.17 (a) Corneal burns induced by a heated copper filament. Note ring of coagulation damage extending
0.5 mm from center of in injury. Topographic changes are evident by stress lines in the cornea seen by retroillumi-
nation in the pupil. (b) Appearance of cornea immediately following focal stromal heating with a non-contact 300
usec, 2.1 um TmHoCr:YAG laser. Large dioptric changes are possible with gross coagulation injury.
annular pattern designed to correct astigmatism (Figure 8.18). With non-contact treatment, the treatment
zone was found to be limited to an inverted cone having an epithelium base of ≈ 300 µm in diameter
and a stromal depth of ≈ 250 µm; the endothelium was spared and the damage to the adjacent stromal
and epithelium tissue was limited to 50 µm of the treatment zone.
8.4.2.3.3 Laser Tissue Welding (Photowelding)
Laser tissue welding involves the use of laser energy to fuse tissue directly or activate biological agents
to bond tissue. It may allow sutureless wound closure and reduction of surgical time, and find application
in corneal, cataract, glaucoma, and vitreoretinal surgery. Laser welding has been applied successfully in
other surgical fields, including vascular, dermatology, urology, otolaryngology, and neurology.40–42 The
pathophysiologic mechanisms of laser tissue welding are not yet fully understood; however, it is postulated
that tissue fusion is due to the denaturation and homogenization of collagen with interdigitation of
individual fibrils that occur when tissue is heated by absorption of laser radiation.43
Welding synthetic collagen lenticules to the cornea with and without the use of solders has been
attempted using a milliwatt CO2 laser.41 Direct welding, either lenticule to lenticule, lenticule to
cornea, or cornea to cornea was not successful without the use of bio-solders. Because the mechanism
for laser tissue welding is probably heat dependent, the short penetration (10 µm) of CO2 laser
radiation tends to overheat and char tissue, producing weak welds. More recently, unique matches
Figure 8.18 (a) Histology of laser photothermal keratoplasty burn in rabbit cornea. The injury is conical in shape
and spares the endothelium. (b) Color-coded map of topographic changes (in diopters) induced in human cadaver
eye treated by LPTK with a TmHoCr:YAG laser operating g at 2.1 µm (300 µs, 1Hz).
between laser and tissue characteristics have been identified that may lead to improvements in tissue
welding.
8.4.2.4 Cataract Surgery with Endo-Laser System
The future goal of cataract surgery is to restore accommodation.44,45 This can be accomplished by removal
of the cataractous lens through a small opening while preserving the lens capsule and its zonular
attachments. The empty lens capsule is then filled with a bio-compatible and optically suitable synthetic
gel, creating a physiologically compatible artificial intraocular lens in situ (Figure 8.19). This procedure
will demand a more precise and less damaging method for cataract removal, which is currently performed
by ultrasonic probes (phacoemulsifiers). Endo laser systems for removing the cataractous lens are the
most promising candidates.
By directing laser energy into a flexible fiber, cataract removal could be performed endoscopically
while minimizing iatrogenic trauma to healthy tissue. Bath demonstrated the successful use of the 308
nm XeCl excimer laser for cataract removal.46,47 Despite several advantages, such as a rapid tissue
removal rate (25 µm/pulse at 6.5 J/cm2), limited thermal damage to adjacent tissue (≤50 µm) and the
availability of inexpensive silica fibers with good transmission properties, the fluorescence induced by
308 nm laser pulses may cause significant retinal damage.48 Potential carcinogenesis and cataractoge-
nesis of UV light (308 nm) to the operator and the patient also raises serious concern about the use
of this wavelength.
Mid-infrared (2.5 µm–3.5 µm) lasers offer an alternative possibility for laser cataract removal. In this
spectral region, the optical penetration depth can be controlled to less than 1 µm by choosing an IR
wavelength that corresponds with the absorption peak of lens tissue (2.9–3.0 µm). The laser energy will
then be confined to a very small volume, preventing photons from reaching other intraocular structures
such as the retina or the corneal endothelium. The short penetration depth of mid-IR laser radiation
also minimizes thermal damage to adjacent tissue, providing the pulse duration is shorter than the thermal
relaxation time of the lens tissue. In addition, laser excitation in the mid-IR region precludes electronic
absorption, eliminating undesirable fluorescence.
Transmitting mid-infrared (2.8 µm–3.1 µm) short laser pulses (≤ 500 ns) through an optical fiber is
a challenging technical problem.49 It may require the use of exotic materials such as zirconium fluoride
(ZnF4) and specially designed probes (Figures 8.20 and 8.21). Efforts to refine this approach and to
integrate a micro irrigation–aspiration system that will be practical for clinical use are under way by a
number of investigators.
Figure 8.19 Phaco-Ersatz. The cataractous lens is removed through a small incision with an integrated endolaser-
irrigation-aspiration system. The capsular bag is retained, allowing injection of a suitable polymer that has optical
and mechanical properties similar to those of the natural lens. Such a procedure may allow restoration of accom-
modation, the ability of the lens to increase its optical power and focus on near objects.
8.4.2.5 Laser Induced Hyperthermia for Ocular Tumors

Ocular tumors include uveal melanoma in adults and retinoblastoma in children. Conventional treat-
ments for these tumors include enucleation, ionizing radiation, external beam or brachytherapy,
photocoagulation and cryotherapy. Tumor-selective hyperthermia (41 °C to 45 °C), and nonselective
hyperthermia, (> 45 °C), are techniques that show potential synergistic effects with other forms of
tumor treatment such as ionizing radiation, chemotherapy and photodynamic therapy.50 Present
methods of producing hyperthermia in ocular tumors include the use of microwaves, radio frequency
waves, and ultrasonic energy sources. The localization of the heating by electromagnetic radiation,
Figure 8.20 Photovaporization of a dense human lens nucleus in vitro with a pulsed HF (350 nm, 2.9 um, 10 Hz)
laser conducted through a shielded ZrF fiber. Practical clinical use will require coupling the laser fiber to an irrigation-
aspiration system to remove lens byproducts and prevent excessive heat build up.
Silica Sheath
ZrF Fiber 480 µm 560 µm
Figure 8.21 Silica clad ZrF fiber used for conducting 2.9 um Hf laser energy for experimental cataract removal.
either transcorneal or transcleral, is very difficult to control and can lead to significant side effects
such as cataract formation.
Near infrared light (700 nm to 1200 nm) can also induce significant tissue heating. Additionally, such
wavelengths can penetrate ocular tissue effectively. Infrared laser wavelengths are scattered in most light-
to medium-pigmented tissue, and absorbed by heavily pigmented tissues. Additionally, the cornea, lens
and vitreous are relatively transparent to infrared wavelengths. These two factors make infrared laser
wavelengths a potential source for producing localized heating in ocular tumors.51
8.4.3 Beam Delivery Systems for Photocoagulation

In ophthalmology, four types of optical delivery systems are currently used. Two are used in the out-
patient setting: (1) A joy-stick-controlled delivery system mounted on the slit-lamp-biomicroscope for
treatment of the patient in the sitting position (Figure 8.13a, b) and (2) an indirect ophthalmoscope
delivery system worn on the surgeon’s head and used with the patient in a 45–70º reclined position. Two
other modalities are used in the operating room with the patient in the supine position: (3) a joy-stick-
controlled system mounted on the binocular operating microscope to treat readily visible and accessible
tissues and, (4) a hand-held fiber optic probe system used for endocular and endo-orbital surgical
treatment (Figure 8.13c).
All types of delivery systems are usually connected to medium power (5 to 8 W) constant wave argon
krypton, dye, pumped dye, constant wave Nd:YAG, and (1 to 3W) AlGaAs diode lasers via a 500 µm
diameter monofilament silica fiber having a numerical aperture of 0.1 to 0.2. Articulated arm delivery
systems are no longer used in ophthalmic photocoagulator designs. The optics for the first three types
of delivery systems are designed to produce a small focal spot on the target (range 50–1000 µm). The
endophotocoagulator probe uses a 300–400 µm core diameter fiber that produces a 500 µm–2 mm spot,
depending on the distance of the probe tip to the tissue. Recently, Rol et al. introduced a series of novel
endoprobes equipped with micro-optics to further reduce the spot diameter and focalize the laser beam
on target.52 This system’s output power is in the range of 100 mW–2W and the energy delivered to target
is 100–10 J. With most delivery systems that are available commercially, the laser power output (0–100%)
and pulse duration (0.1 to 1 sec, in 0.1 sec increments) are preset on the control panel by the surgeon
who activates the laser using a foot-pedal switch. For safety, several interlocks are provided by the
manufacturer in accordance with laser safety regulations.
To prevent contamination and permit mobility of surgical equipment, single-phase (110 or 220 VAC)
and air cooled laser operation is preferred in the clinic and almost mandatory in the aseptic operating
room. As with many medical instruments, miniaturization of the laser and delivery systems are prereq-
uisites for ophthalmological use.
The use of subminiaturized endoscopes designed for intraocular photocoagulation of the ciliary body
and anterior retina under visual control have been introduced recently.45 Thus far, this technique has
been restricted to in vitro and animal studies.
For other ophthalmic surgical procedures, subminiature fiberoptic endoscopes provide the surgeon
with a good view and illumination and lead to more precise surgical procedures than the more conven-
tional “open-sky” surgical techniques. At the present time, endolaser surgical procedures require different
types of fibers for tissue ablation, coagulation, shrinkage, welding, and sensitization of tissue, and
illuminating and imaging of the surgical field. In the future, these different functions may be integrated
into a single fiberoptic delivery system, facilitating surgeon control.
Diode lasers, introduced in 1987, are the newest photocoagulators.53 These lasers have the advantage
of greatly decreased size, cost, and reduced maintenance. Presently, AlGaAs diode lasers emitting at 805
nm with output power 1–3 W have been used for the treatment of retinal vascular disease. Clinical
advantages of the diode laser include minimal absorption and scattering in cataract or mild vitreous
hemorrhage compared with the argon green wavelength and low absorption by intraretinal hemorrhage.
Excellent laser penetration through macular edema and serous retinal thickening have also been dem-
onstrated. Technical disadvantages of the diode laser include a limited power output and a more divergent
beam cone angle than the argon and krypton lasers used presently. Because semiconductor laser tech-
nology has advanced rapidly in recent years, the future outlook for semiconductor lasers is very promising.
Diode lasers and diode-pumped solid state lasers with multi-watt power output at wavelengths that are
desirable for various clinical applications will be available to replace the more expensive and bulky argon,
krypton, and dye lasers for ophthalmic applications. Q-switched and mode-locked diode lasers with
sufficient energy density in the nanosecond to picosecond range may also replace the Nd:YAG laser as a
photodisruptor for iridectomy, trabeculoplasty, and cyclophotocoagulation.
8.5 Photodisruptive Laser Applications
8.5.1 Photodisruptive Laser–Tissue Interaction

A disadvantage to laser applications utilizing photothermal mechanisms is that the laser wavelength and
absorption properties of the tissue must be carefully matched. Tissues transparent to the incident wave-
length will be unaffected.
In the late 1970s, short pulsed 1064 nm Nd:YAG lasers (≈ 30 ps for mode-locked and ≈ 10 ns for Q-
switched) were introduced to create optical breakdown and photodisruption of ocular tissues. The
Nd:YAG photodisruptors are now in common clinical use. The laser beam is brought through a high
numerical aperture beam expander–focusing lens system (20–40 cone angle) to a sharp focus (≈ 10–50
µm diameter) to achieve extremely high irradiances (108–1011 W/cm2) such that ionization of molecules
occurs at the target. The irradiated material disintegrates into plasma, a collection of ions and electrons,
that reaches peak temperatures of ≥104 ºC. Shock waves emanating from the site of plasma formation
cause mechanical disruption of structures adjacent to the site of optical breakdown. Because optical
breakdown is a threshold phenomenon that depends on the strength of the electrical field, photodisrup-
tion is not dependent on absorption of the wavelength by the target tissue, and photodisruptors can be
used in tissues that are transparent to the incident laser wavelength. For optical breakdown to occur, it
is only necessary for the photodisrupting laser delivery system to achieve the required high irradiance
within a small volume of tissue.
8.5.2 Photodisruptor Applications

Photodisruptor laser applications are useful for cutting, incising, or vaporizing intraocular tissues. Table
8.2 summarizes the clinical and research applications for photodisruptors in ophthalmology.
Table 8.2 Photodisruptor Laser Applications

Procedure Disease Laser/Wavelength Rationale Comment
Posterior Capsular Nd:YAG-106-1 Photodisruption Widespread use —
capsulotomy opacification after allows small replaced need for
cataract surgery opening to be intraocular
created without procedure
IOL damage
Lysis of vitreous After trauma, Nd:YAG-106-1 Adhesions can be
strands surgery severed without
entering eye
Peripheral iridotomy Refractive errors Nd:YAG-106-1 Removing tissue Experimental
2nd harmonic-562 from stroma
changes corneal
refractive power
Endolaser vitreous Diabetes, trauma Nd:YAG-106-1 non-contact cutting Experimental
surgery Er:YAG-2.9 µm
8.5.2.1 Posterior Capsulotomy

By far the most common and successful photodisruptor application is the creation of a posterior capsu-
lotomy with a Nd:YAG laser, performed several months or years following extracapsular cataract surgery
when the posterior capsule, left intact by the surgeon to support a plastic intraocular lens (IOL), opacifies
due to proliferation of retained lens epithelial cells.54 The patient, who experienced improvement of vision
following surgery, suffers slowly declining vision as the capsule opacifies. The surgeon uses the Nd:YAG
photodisruptor to create a central opening in the capsule, restoring vision. The surgeon must be careful
to focus on or slightly behind the posterior capsule, or undesirable pitting or cracking of the polymethyl
methacrylate intraocular lens can occur.
The use of the Nd:YAG laser in ophthalmology was simultaneously introduced by glaucoma specialist
Franz Fankhauser in Switzerland (Q-switched) and cataract specialist Danielle Aaron-Rosa in France
(mode-locked) in 1980. Two years later, the first international congress dedicated to the theory and
clinical use of these lasers was held in Munich and the technique gained rapid acceptance thereafter.
8.5.2.2 Vitreo-Retinal Surgery
The Nd:YAG photodisruptor has also been used for cutting vitreous strands in the vitreous cavity and
anterior chamber, creating peripheral iridectomies, severing trapped IOL haptic loops, and cutting
sutures. These lasers allow outpatient surgical treatment and have reduced postoperative complications
such as inflammation and infections associated with intraocular surgical procedures.
In 1980, Parel and Fankhauser designed a 1 mm-diameter Q-switched Nd:YAG laser miniature
endoprobe delivery system for severing vitreous and proliferative retinal membranes (Figure 8.22). In
these early experiments, Fankhauser’s group and ours failed to transmit short 10 nsec pulses through
the silica fibers that were available at the time. After designing a novel laser-to-fiber focusing mechanism,
and making repetitive attempts, Rol and Fankhauser were successful in transmitting 10 ns (Q-switched),
and 250 µs to 20 ms (free-running) Nd:YAG pulses of sufficient energy for photodisruption and photo-
coagulation of intraocular tissues using high purity silica fibers. More recently, Margolis et al. successfully
photoablated intraocular proliferative membranes using a free-running Er:YAG coupled to a bare ZrF4
fiber optic.55 However, hygroscopic, mechanical and optical problems, including laser beam spatial
stability, remain formidable and further research is required in fiber and laser technology before a
clinically usable endolaser photodisruptor instrument is available.
8.5.2.3 Intrastromal Corneal Photodisruption
Recently, picosecond and nanosecond photodisruptors have been designed for use in corneal surgery.
Focused in the middle portion of the central cornea, the high repetition rate (l03–104 Hz) pulsed laser
beam is scanned parallel to the corneal plane. Photodisruption transforms the thin lamellae of the corneal
stroma into gas that, after absorption, may change the outer corneal curvature, resulting in a change in
the eye’s refractive power. The theoretical advantage to this approach is that the epithelium and Bowman’s
layer are left intact and, therefore, the unpredictable refractive effect of corneal wound healing may be
minimized. In addition, this technique is thought to minimize thermal damage to the collagen lamellae
adjacent to the photodisrupted zone. Two systems are presently under development; a frequency doubled
(562 nm) mode locked (30 ps) Nd:YAG (562 nm, 30 ps) laser and a Q-switched 1064 nm (5 ns) Nd:YAG.
Both use very high numerical aperture aberration-free optical systems to minimize the volume of pho-
todisrupted tissue. Although this approach is intriguing in theory, thus far no data has been published
indicating intrastromal photodisruption can alter the refractive status of the eye effectively or predictably.
Basic laboratory and clinical research with this technique is continuing.
8.5.3 Photodisruptor Delivery Systems

Because it is technically difficult to transmit Q-switched laser pulses and impossible to transmit mode-
locked laser pulses with sufficient energy for tissue photodisruption through optical fibers, the optical
delivery of the photodisruptor laser beam is accomplished via sets of high quality mirrors and high
precision articulated arms. The laser optics are normally integrated physically in a conventional slit-lamp
biomicroscope or operating microscope in a fashion similar to that shown for the photocoagulating
instruments (see Figure 8.12). Because focusing of the photodisrupting laser beam on target is very
critical, a set of two or more confocal He-Ne laser beams are focused on the target tissue while observing
under magnification (20–40 ×). To facilitate focusing, the He-Ne laser beams are made to rotate or
synchronously alternate so as to produce a single stable spot when focus on target is reached. To minimize
the optical aberrations produced by the cornea and off-axis treatment, special contact lenses are normally
used. These lenses further increase the numerical aperture of the optical delivery system and therefore
minimize the spot diameter and improve overall efficiency. With such lenses, capsulotomy can be per-
formed with as little as 0.5 mJ using a 10 ns Q-switched Nd:YAG laser. The systems designed for corneal
intrastromal ablation also incorporate an eye-tracking device to achieve the high spatial resolution
necessary for this procedure. These systems can also be used for conventional photodisruptor applications
such as capsulotomies and iridotomies.
8.6 Photochemical Laser Applications: Photoablation

and Photodynamic Therapy
8.6.1 Photoablation Mechanism

A new form of laser–tissue interaction was discovered in 1983 by Trokel and Srinivasan, who were working
at IBM.56 Srinivasan was studying the far-UV (193 nm) argon fluoride excimer laser for photoetching
applications when Trokel, an ophthalmologist, noted that corneal tissue could be removed discretely and
precisely without any histological evidence of thermal damage to the adjacent corneal tissue. Trokel
recognized the potential of the excimer laser to offer a new approach to corneal surgery: corneal sculpting.
Photoablation occurs because the cornea has an extremely high absorption coefficient at 193 nm, and
the photons at 193 nm are highly energetic, possessing more energy than the carbon–carbon bonds
interlinking the protein molecules of the cornea. Consequently, 193 nm laser photons rupture, intermo-
lecular bonds and a discrete volume of corneal tissue is removed with each pulse of the laser
Figure 8.22 Endo-laser probe. An aspheric microlens focuses the laser beam above the vitreous fluid aspiration
port. Endoscopic illumination is provided by a conventional coaxial 12 µm fiber bundle. The hook is designed to
lift and separate the membranes to be cut from the delicate retinal tissue and provides a shield that prevents laser
radiation and plasma-induced secondary radiation from reaching healthy tissues. An electromechanical beam
switcher (Nd:YAG to argon) located in the remote console allowed intraoperative photocoagulation of intraocular
bleeding vessels.
(Figure8.23).57,58 The depth of the ablation is dependent on the radiant exposure and typically varies
from 0.1 to 0.5 µm per pulse at a radiant exposure of 50 to 250 mJ/cm2 (Figure 8.24).59
The laser–corneal tissue interactions of other far-UV excimer wavelengths including 157, 248, 308,
and 351 nm have been investigated, and the 193 nm wavelength generated by the argon fluoride excimer
has been found to achieve the best quality corneal excisions for clinical use (Figure 8.25). The 157 nm
produced by the fluorine dimer produced similar-quality excisions,60 but its use is not practical clinically
because of high atmospheric absorption at this wavelength.
Initially, it was suggested to use the excimer laser as a “laser scalpel” for corneal surgery in operations
such as radial keratotomy.61 However, the excimer laser was found to be a poor replacement for a cutting
scalpel, because the laser removes rather than cutting a defined volume of tissue. Even with high quality
aberration-free optics, excisions smaller than 60 µm were difficult to produce due to multimode operation
and poor beam homogeneity. Clinical attempts to use the excimer laser with a set of linear slit-masks
for radial keratotomy resulted in even wider (250–300 µm) excisions (Figure 8.26). Because of the wide
excisions and poor corneal stromal wound healing that followed after deep laser excisions, the use of the
excimer laser for this type of surgery has generally been abandoned.
The most exciting application of the excimer laser was found to be its ability to sculpt or reshape the
outer de-epithelialized surface of the cornea to correct refractive errors. This relatively new surgical
procedure was named photorefractive keratectomy (PRK) by its developers (Figure 8.27).62 PRK underwent
considerable scrutiny because treatment of the central cornea — the most important image-forming
element of the eye — was involved. Early animal studies were encouraging,63,64 and as the laser and delivery
system technology matured, confidence was gained that sighted human eyes could undergo excimer laser
corneal sculpting without vision threatening complications. The first sighted-eye procedure was performed
in 1988,65 and, since that time, several thousand have been performed in Europe and several hundred in
the USA as part of a carefully controlled FDA study conducted by three manufacturers: Summit Technology
(Watertown, Massachusetts), Taunton Technologies, and VisX (Sunnyvale, California), (the latter two
companies have recently merged). Present data indicate that PRK appears to be a safe and effective method
for reducing myopia up to 6 diopters with few side effects or complications.66–69 Treatment of myopia
greater than six diopters has been less successful due to regression of the refractive effect caused by corneal
wound healing.70 To date, attempts to steepen the cornea to treat hyperopia with the excimer laser have
Figure 8.23 193 nm argon fluoride laser pulses result in a unique photochemical interaction with the cornea
termed photoablation. The laser pulse (a) is highly absorbed in the first several microns of corneal tissue, primarily
by protein chromophores (b). Because the 193 nm photons have energy in excess of the carbon-carbon intermolecular
bonds of corneal tissue, the bonds are photoelectrically decoupled, resulting in the ejection of many small fragments
from the surface (c). The photoablation process is completed after 15 ns, leaving clean, precise edges with only 0.3
um of adjacent tissue alteration.
been unsuccessful because regeneration of the corneal stroma and thickening of the epithelium cause the
curvature to return to its preoperative shape, negating the refractive effects of the procedure.
Now approved for general use, the laser may offer an alternative to glasses and contact lenses for
millions of myopic individuals, and may represent the most widespread medical application for a laser.71
Longer term studies to assess the overall predictability, stability, and safety of the procedure are under way.
Although the excimer laser is entering clinical use, this gas laser has several disadvantages. The apparatus
is large and bulky, complicating its use in the clinical environment. Fluorine gas is toxic (ED50 = 0.3 ppm)
and requires special safety considerations. Therefore, a solid state laser source in the far UV is desirable.
Ren et al. have demonstrated generation of 213 nm from a frequency quintupled Nd:YAG laser using
nonlinear frequency multiplying crystals (Figure 8.28).72 They demonstrated high quality photoablation
ABLATION DEPTH/PULSE
1.0
0.8
(mM/pulse)
0.6
0.4
0.2
0
0 0 0 0 0 0 0 0 0
10 20 30 40 50 60 70 80 90
RADIANT EXPOSURE
(mJ/cm2)
Figure 8.24 Ablation depth of the human cornea per pulse vs. radiant exposure with the 193 nm excimer laser.
Tissue ablation rate will vary with the hydration of the cornea.
Figure 8.25 High quality graded corneal excisions produced with the 193 nm excimer laser using the Hanna-IBM
moving slit delivery system. Single epithelial cells (Ep) have been cleaved with the laser. Note the recontouring of
Bowman’s layer (BM) possible with the laser (S = corneal stroma).
of corneal tissue, similar to 193 nm excimer excisions with the 213 nm wavelength. Whether the frequency
multiplied solid state lasers can become practical clinically remains to be seen.
8.6.2 Delivery Systems for Photoablative Lasers

Several beam delivery systems have been developed to homogenize, shape, transmit, and focus the ArF
excimer laser for corneal ablation. Methods of beam homogenization include moving lens arrays and
cylindrical optics. Beam shaping strategies include the use of one or several apertures whose dimension,
shape and position relative to the corneal apex are normally controlled by a computer. Rotating wheels
that contain apertures of several shapes and sizes,73 expanding or constricting iris-diaphragms,74–76 math-
ematically defined slits, and moving perforated masks have been used to regulate the energy delivered to
Linear
Excision
Figure 8.26 Deep linear corneal excisions produced with a 193 nm excimer laser and mask system. The excisions
filled with an epithelial plug and were found to heal poorly, resulting in abandonment of this approach.
Photoablative
Keratectomy for
Myopia
Figure 8.27 Photorefractive keratectomy (PRK) for myopia with the 193 nm excimer laser. The stromal surface
of the cornea is recontoured with the laser following removal of the epithelium by the surgeon. Following reepithe-
lialization, the anterior corneal curvature is flatter, reducing myopia. Preliminary evidence indicates that this tech-
nique is safe and effective for reducing myopia up to 6 diopters.
the cornea in optical delivery systems. A different approach utilizes a preshaped photoablatable synthetic
mask positioned immediately above the corneal surface. A uniform irradiation beam pattern is required
with this technique. All of these concepts are based on surface ablation using commercially available
excimer laser systems with quasi-rectangular beam patterns (usually 25 mm × 7 mm) and low repetition
rates of 10 to 20 Hz (Figure 8.28). The low repetition rates require that these systems treat relatively large
areas of the cornea with each pulse to complete the treatment within a reasonable clinical time frame
(30–60 seconds). Therefore, with the exception of the synthetic photoablatable mask, the present delivery
systems are limited to creating simple spherical or sphero-cylindrical optical corrections.
A laser diode pumped, rapidly pulsed (KHz range) UV solid state laser (frequency-quintupled Nd:YAG
laser emitting at 213 nm) might offer an alternative laser source for corneal sculpting. State-of-the-art scanning
technology could be used to “paint” the cornea in any desired pattern, which may facilitate aspheric and
Ablatable
Rotating Disc
Diaphragm
Scanning
Figure 8.28 Different methods of shaping the excimer laser beam include an ablatable polymer mask, computer
controlled diaphragms, rotating discs, and movable elliptical masks.
spatially irregular corneal sculpting.77 However, multiple technical challenges that face this technology, includ-
ing low crystal damage thresholds, small spot sizes, and unstable power outputs, must first be overcome.
8.6.3 Photodynamic Laser Applications

Photodynamic therapy (PDT) utilizes a selective laser wavelength to activate a photosensitizing agent
that, in turn, causes damage or destruction to malignant or abnormal tissue. A photosensitizer is a
molecule or compound that, when excited by an appropriate wavelength, can transfer energy to oxygen
in tissue to produce singlet oxygen, or can dissociate to produce free radicals. Phototoxicity results from
the biochemical interaction of tissues with the singlet oxygen and radicals and is a function of the
photosensitizer’s quantum efficiency and absorption spectra, its concentration in tissue and the wave-
length and fluence of the irradiation.
Only recently has photochemical reaction with cellular and vascular structures been introduced as a
means of therapy.78 Dougherty recognized the potential of di-hematoporphyrins ether (DHE) in the
treatment of malignancies. Following intravenous injection, these photosensitizing agents remain longer
in rapidly proliferating cancerous tissues, and allow a localized phototoxic effect to be induced by use of
adequate photo irradiation. Dougherty developed this procedure, which he named DHE photodynamic
therapy (DHE-PDT), more than 20 years ago. The treatment method consisted of injecting 25 µg/ml of
DHE (Photofrin II) per Kg body weight followed by a 100–250 J/cm2 irradiation at 630 nm with a dye
laser 48 hours after injection. Irradiation was performed near a DHE absorption peak at 630 nm because
this wavelength has a deeper tissue penetration than DHE’s other absorption peaks. For photoactivation
of DHE, a constant wave argon pumped 630 dye laser with 1–2 W output is normally used. Postoperative
complications of photodynamic therapy include long lasting (up to 3 weeks) hyper-photosensitization
in certain patients. Several centers are investigating the use of other photosensitizers, including phtallo-
cyanides, that can be excited at longer wavelengths (also see Chapters 6 and 10).
Apart from the treatment of basal cell carcinomas of the face and eyelids, ophthalmic applications of
photodynamic therapy have been limited to attempts to treat ocular melanomas and conjunctival carci-
noma.79 Because HDP-PDT has failed to demonstrate efficacy to date, several authors are investigating
the use of other photosensitizers, including rose-bengal,81 and phtallocyanide.53 In addition, other authors
are investigating photodynamic therapy for the treatment of proliferating vascular disorders of the
cornea81 and retina,82 the treatment of fungal infections,84 and the treatment of proliferative lens epithe-
lium following cataract surgery.84–88 As the later type of treatment is superficial in nature, the constant
wave argon 514.5 nm wavelength has also been used successfully. However, because the peak absorption
of rose bengal is highest at 562 nm, a solid state laser operating near this wavelength would facilitate
ophthalmic applications.
The laser delivery systems used for PDT are similar optically to those shown in Figure 8.12a, b; a
timing mechanism connected to the laser foot-switch control is used to calculate the total radiant dose
given to the patient. Clinicians have been plagued by technical problems associated with the argon
pumped dye laser. Present systems have not incorporated means to adjust and control the output
wavelength in the laser console and, in many instances, irradiation is performed with an off-peak
wavelength. Furthermore, the laser output power tends to fluctuate during treatment. These problems
lead to sub-threshold dose and inadequate treatment. In addition, dye laser breakdown is too common
in the clinical setting. Because DHE is injected 48 h prior to treatment, instrumentation breakdown
might cause serious medical complications because the clinician must wait 5 days to several weeks for
drug clearance in hypersensitive patients before repeating treatment. A solid-state laser providing an
output of 1 W at the wavelengths used for photodynamic therapy would be of tremendous clinical value
and facilitate the investigation of this new therapeutic modality.
8.7 Diagnostic Laser Applications

The high radiance, monochromaticity, and spatial and temporal coherence make the laser a unique light
probe for noninvasive diagnostic applications in ophthalmology. The information contained in laser light
reflected or scattered by intraocular structures can be detected and analyzed for diagnostic purposes. In
general, light scattering is a phenomenon inversely proportional to the fourth power of the wavelength,
thus, blue light is scattered more than red light.
8.7.1 Visual Acuity Measurement

In this application, the spatial coherence of lasers is used to form interference fringes on the retina. These
are dark lines with variable spacing and orientation whose formation is largely insensitive to the optical
clarity of the intervening media. The retina’s functional health may be evaluated even when it is obscured
by a cataract or other cloudy media existing in the eye. The spacing of the fringes is varied by adjusting
the angle of two interference beams and the patient indicates whether they are visible, facilitating
prediction of the patient’s acuity after cataract surgery. Fringe contrast can also be varied by adjusting
the relative intensity of two laser beams, yielding the retinal modulation transfer function (MTF) —
termed the contrast sensitivity function when plotted reciprocally. The light source used for interferom-
etry is generally a He-Ne laser, and the retinal irradiance is in the range of 1 µW/cm2. This technology
has widespread clinical use today and is very helpful to the ophthalmologist in deciding whether cataract
surgery will be of benefit to the patient.
8.7.2 Laser Doppler Velocimeter

In laser Doppler velocimetry (LDV), laser light scattered by moving blood cells is shifted in frequency
and is used to measure the rate of blood flow in the veins and arteries of the retina. In this application,
the temporal coherence of lasers is used. The light scattered at a particular angle by the flowing particles,
as well as the light scattered from the vessel wall, is collected by a photodectector. The light scattered
from the vessel wall is unshifted in frequency and thus acts as the reference beam. Interference between
the reference beam and the scattered light can be used to generate a difference in frequency that is directly
related to the maximum red blood cell velocity at the measurement site. In this application, it is important
to keep a well defined spot on the site to be measured and to know reliably the angle between the incident
and scattered light.
LDV is a research tool that is providing important information to scientists studying retinal blood flow.
8.7.3 Scanning Laser Ophthalmoscope

The scanning laser ophthalmoscope (SLO) was developed by Webb for viewing the retina and its sup-
porting structures including blood vessels, nerve bundles, and underlying layers.89–91 In scanning laser
ophthalmoscopy, a laser beam about 1 mm in diameter at the eye’s pupil is focused to a 10-µm spot on
the retina. The beam is scanned over the retina without changing its location at the pupil (it “pivots”
within the entrance pupil). At any instant, only a 10-µm spot on the retina is illuminated, and that instant
may last only 100 ns. Light in the illuminating spot may be absorbed or scattered. If absorbed, the detector
at that instant records no response, and the display shows black. If the light is scattered, however, some
of it will reach the detector, the detector records a voltage and the display shows a spot with the
corresponding gray level intensity.
The SLO uses a highly collimated laser beam for illumination, thus requiring a smaller entrance
aperture and less sensitive collection optics than conventional optical imaging systems. The image
detected by the SLO is temporally rather than spatially coded, as with an indirect ophthalmoscope.
The SLO has opened a new dimension in psychophysics, because it allows direct observation of the
retinal location on which the stimulus is presented. Future applications include use as a therapeutic
device (photocoagulator) and a retinal eye tracker.
8.7.4 Spectroscopic Diagnosis of Ocular Diseases

Spectroscopic techniques such as fluorescence spectroscopy and Raman spectroscopy are under inves-
tigation as noninvasive means to detect various abnormal states of ocular tissues and ocular diseases.
Much of the research effort has been concentrated on detection of early cataract formation and certain
vitreous disorders.
The transparency of the lens changes normally with age, and sometimes degenerates entirely with the
formation of a cataract. Laser Raman spectroscopy has been employed as a noninvasive probe to study
the metabolic and structural features of the lens.92–94 With newly developed near infrared (NIR) Fourier
transform (FT) Raman spectroscopy, fluorescence interferences from heavily pigmented ocular lenses
can be eliminated and spectroscopic information can be remotely acquired via fiberoptic probe and
analyzed in real time.95 An automated laser-scanning-microbeam system has been developed for the
fluorescence and Raman imaging of the human lens. Age-related fluorophor conversion (from blue to
green) and intensity change in spatial distribution have been identified.96,97 This spectroscopic informa-
tion has added to our understanding of the biochemical changes associated with cataract formation.
8.8 Summary
During the past two decades, laser technology has revolutionized many aspects of ophthalmic surgery.
Millions of patients have benefited from the restoration or preservation of vision from laser treatment.
The use of lasers to successfully treat retinal diseases, glaucoma, and capsular opacification following
cataract surgery is now the standard of care. The end result of the interactions among basic scientists,
clinicians, and industry that have produced this revolution has markedly improved the ability of oph-
thalmologists to help their patients.
Nevertheless, potential for abuse and exploitation of laser technology exists and should be resisted.
The use of expensive lasers should be limited to procedures that they can do uniquely or do more
effectively than simpler, inexpensive technology. Use of lasers for dubious indications for a medical
marketing advantage not only escalates the cost of health care unnecessarily, but further elevates the
public’s unrealistic expectation of what laser technology can actually deliver.
Still, the future of laser technology in ophthalmology looks very bright. The excimer laser may offer
improvements in the safety and efficacy of refractive surgery, and could give millions of myopic individuals
a viable alternative to dependence on glasses and contact lenses for clear vision. Like parallel advances
in computer technology, existing lasers are expected to become smaller, more capable, and less expensive
as solid state laser technology matures.
Acknowledgments
We are grateful to Drs. Edward W.D. Norton, Victor Curtin, Gary Margules, Takashi Yokura, William Q.
Jeffers, Michael Berry, Shiro Takizawa, Gerard Kervick, Tom Patrick, Pascal Rol, Michael T. Gill, Ms.
Mary Ann Taylor, and Ms. Barbara French for continuous scientific and moral support.
Supported in part by: Research to Prevent Blindness, Inc. by P30 EY06360 (a NIH Departmental Core
Grant), Florida Lion’s Eye Bank, Topcon Research Institute, Helena Rubinstein Foundation, NEI
#EY02180 and EY07803-01, Florida High Technology and Industry Council, Miami Veterans Adminis-
tration Medical Center, T32 EY07092 NIH Training Grant, and P30 EY06360 Departmental Core Grant
from NIH.
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9
Cardiovascular
Applications of Lasers
9.1 Introduction ....................................................................... 247

9.2 History ............................................................................... 248
9.3 Anatomy and Pathology .................................................... 249
9.4 Physics ................................................................................. 251
9.5 Angioplasty (with Mark H. Wholey, M.D.) ..................... 262
9.6 Transmyocardial Laser Revascularization (TMLR) ......... 268
(with Osvaldo J. Yano, M.D, and
Mehmet C. Oz, M.D.) ........................................................ 271
Valvulotomy and Debridement • Myotomy, Myomectomy, and
Myoplasty • Septostomy • Endarterectomy • Vascular
Anastomoses: Laser Welding in the Cardiovascular System •
Jordan D. Haller, M.D Aids to Welding: Tissue Solders • Clinical Examples of Tissue
Columbia University School
Welding
of Public Health
References ...................................................................................... 278
9.1 Introduction
This chapter will review the cardiovascular applications of lasers from their initial experimental use
shortly after the invention of the laser, through a period of unrealistic expectation and enthusiasm, and
to the present period of skepticism. The medical and scientific bases for the application of lasers are
summarized so that the causes for failure as well as the potential for benefit of cardiovascular uses can
be understood in developmental perspective. Medical terms and conditions are explained for the physical
scientist. Laser–tissue interaction is stressed. Engineering and ancillary physical science topics such as
fiber optics are omitted for lack of space.
In the course of their medical practice, ophthalmologists and dermatologists regularly deal with the
effects of light on patients’ health, so it is not surprising that they were the first medical specialists to
turn to lasers as potential diagnostic and therapeutic aids very shortly after the invention of the laser.
The cardiovascular system, however, is not exposed to light, and cardiovascular medical specialists are
not usually familiar with the special biologic effects of light and the physical and chemical properties
and limitations of various lasers. Access to the cardiovascular system poses an additional challenge. Not
only does the cardiovascular physician usually not have a background in the physical-chemical properties
of light, but, to be able to treat the disease process, additional technologies such as fiber optics and
catheter delivery must be utilized and understood to bring the laser light into the cardiovascular system.
Lack of fundamental scientific knowledge led to unnecessary confusion and allowed some cardiovascular
investigators to pursue impossible goals, while extravagant marketing claims were made for laser angio-
plasty. The initial unrealistic expectations for laser use in the cardiovascular system have largely been
0-8493-1146-2/02/$0.00+$1.50
replaced by more objectivity. There remains, however, great need for the cardiovascular specialist to
understand the limits as well as the opportunities posed by the physical science. The contributions of
physical scientists to this educational process are essential and cannot be over estimated.
9.2 History
The first recorded series of laser experiments on the cardiovascular system were by McGuff et al. in
19631 This group exposed fresh postmortem atherosclerotic plaques to short pulses (0.5 to 3.0 ms)
of a ruby laser at 10 to 13 J and noted that “the athero-matous plaques were affected more than the
calcific plaques.” McGuff concentrated on the effect of laser energy on tumors and summarized his
work with the cardiovascular system in 1966,2 observing, “Calcific plaques appeared to be more
resistant to the laser energy and were grossly little damaged by a 10 joule burst.” He noted, as had
physical scientists working with inert materials, that shock waves of supersonic velocity spread
through adjacent biologic tissues, but concluded (erroneously, as results would later show) that they
were not of sufficient strength to cause damage to non-ocular tissues. McGuff also noted that bone,
which contains inorganic calcium salts similar to that which occurs in atherosclerotic plaque, was
“more resistant to laser energy than any other biologic tissue.”
Unfortunately, these basic observations went largely unnoticed and subsequently unheeded by
later cardiovascular investigators. Inappropriate applications of laser energy, mainly in various forms
of laser angioplasty caused needless pain and suffering, while millions of dollars were spent and
ultimately lost in failed business investments. The results of laser angioplasty are reviewed in Section
8.5.
Yahr, Strully and Hurwitt reported in 1964 on “… the enhancement of laser action by selective stains
which affect the relative transparency of tissue.”3 Subsequent efforts to enhance or tag tissue to obtain
selective absorption of laser energy by certain cardiovascular chromophores are discussed in the appro-
priate sections. This group also created the first laser-welded vascular anastomoses and reported addi-
tional vascular studies with the Nd:YAG and CO2 lasers in 1966.4
The next reports on the use of a laser to aid in vascular anastomoses were by Morris and Carter at
the Orthopedic Research Society in 19805 and by Jain and Gorisch in 1979.6 Both groups described the
use of Nd:YAG to weld small-diameter vessels in experimental animals.6 Although laser-welded anasto-
moses of blood vessels have subsequently been shown to be possible and may even have some theoretical
advantages over the traditional suture-created anastomoses, the additional time required has mitigated
against their use. This topic is reviewed in Section 9.7.5.
In 1974, Arapov reported that, under direct vision, he had used a laser to incise a pulmonary
valve whose leaflets were fused in the closed position. 7 However, his paper was published in Russian
and went largely unnoticed. More recently, however, lasers have been used in combination with
balloons to open narrowed (stenotic) valves and, in fact, valves congenitally fused closed in newborns
(neonates).8
The era of “laser angioplasty” was ushered in by the report of Macruz et al. in January 1980,9 in
which they described the experimental use of an argon ion laser to remove atherosclerotic plaque.
This paper was written in Portuguese and published in Brazil, but it contained an English abstract
that was widely circulated.10 In May 1988, at a meeting in China, Choy10 described a catheter to
deliver laser energy to open blood vessels that were occluded by either atherosclerosis or thrombosis.
He had filed a patent for this laser catheter delivery system in 1978, but this was not disclosed until
June, 1980.
Macruz and associates reported on additional experiments with an argon laser at the Fourth Congress
of the International Society for Laser Surgery in Tokyo (November, 1981). They also described the use
of a laser to weld closed incisions that had been made in arteries (arteriotomies) and to create arterio-
venous and arterio-arterial anastomoses (functioning connections). Using fiberoptic catheter delivery,
they reported the performance of laser catheter pulmonary valvulotomy and angioplasty in living dogs
at the October 1981 American Heart Association Meeting.11
Cardiovascular Applications of Lasers 249
FIGURE 9.1 Vascular anatomy and access sites for endovascular diagnosis (angiography) and treatment by percu-
taneous transluminal angioplasty (PTA), including LASER angioplasty
9.3 Anatomy and Pathology

An overview of the cardiovascular system is shown schematically in Figure 9.1 The artery at the elbow,
the brachial, and the artery at the groin, the femoral, are large and superficial. Their pulses can be felt
easily so they can be entered relatively easily by a needle puncture without a surgical incision. The
Seldinger technique,12 by making access possible to any artery in the body, as well as to the heart itself,
including its individual coronary arteries, permitted the development of the diagnostic era of angiography,
which, in turn, has led to the current endovascular therapeutic era. After placing a hollow needle into
either the brachial or femoral artery, a flexible guide wire is inserted through the lumen of the needle.
Finger pressure is held over the puncture site until a slightly larger-diameter flexible plastic catheter can
be threaded over the guide wire. The larger catheter minimizes blood leakage from the smaller needle
hole. The catheter is then guided under fluoroscopy into the vessel that is to be studied or treated. The
guide wire is removed, radiographic contrast material is injected to visualize the area and a series of x-
ray films are taken, on video or on film, with a rapid cassette changer. The diagnostic catheter can then
be removed and replaced by the laser delivery catheter or a balloon catheter or, in fact, any endoluminal
mechanical device to perform the percutaneous transluminal angioplasty (PTA), PTCA for coronary
arteries, or PTLA when the laser is used.
The anatomy of the heart is shown in Figures 9.2 and9.3. Blood enters the right atrium (RA) of the
heart from the superior and inferior vena cavae (SVC, IVC). It passes through the tricuspid valve into
the right ventricle (RV) and from there is pumped out into the pulmonary arteries (PA) to the lungs to
be oxygenated. The entire pulmonary artery, from its origin in the right ventricle to its bifurcation into
right and left branches, has been removed in Figure 9.2 to show the more posterior ventricular septum
(IVS) and aortic valve. Oxygenated blood returns from the lungs via the pulmonary veins (PV) into the
left atrium (LA) and passes through the mitral valve into the left ventricle (LV). This is the thickest, most
muscular, chamber of the heart. The left ventricle pumps blood through the aortic valve into the arterial
FIGURE 9.2 Simplified drawing of the

heart. (From The Artificial Heart: Proto-
types, Policies, and Patients, J.R. Hogness
and M. VanAntwerp, Eds., National
Academy Press, Washington, D.C., 1991).
system to the entire body. After passing through the individual organ and tissue capillaries, the deoxy-
genated blood returns through the veins to the vena cavae and the cycle is repeated. Note the origin of
the coronary arteries at the base of the aorta, just above the aortic valve. Although the heart is filled with
the blood it pumps to the entire body, none of this blood can be used to nourish the heart, which receives
its own blood supply through coronary arteries. See Section 9.6 for the Transmyocardial Laser Revascu-
larization studies being done to try to take advantage of connections between the ventricular chambers
and small vessels within the ventricular walls.
Several types of congenital deformities prevent the flow of blood through the heart. Death of the
newborn will occur quickly unless an opening can be made in the thin interatrial septum (septostomy)
or unless a malformed intracardiac valve can be opened (a valvulotomy — note: the term valvotomy is
sometimes used interchangeably; grammarians have been debating which term is correct for several
centuries and have not reached a conclusion; priority of first use seems to favor “valvulotomy”). Abnormal
thickening or hypertrophy of any part of the heart muscle, most commonly the left ventricle, may be
treated by incision of the muscle fibers (myomotomy or myotomy) or by excision (myomectomy). In all
of these conditions, the tissue is either fibrous or muscular and no inorganic calcium particles are
interspersed. The tissue is pure and homogeneously organic. Laser treatment of many of these conditions
has been attempted and is discussed in Section 9.7.
The most common acquired diseases of the cardiovascular system are due to aging and deterioration,
leading to atherosclerosis in blood vessels and stenosis and insufficiency of the aortic and mitral valves.
These are complex biochemical and metabolic conditions and, once they are severe enough to cause
symptoms, they always contain inorganic crystals mixed with the original basic organic tissues. They
pose the greatest therapeutic challenge to physician and scientist alike. Laser angioplasty is discussed in
Section 9.5.
Figure 9.3 shows the three-dimensional relationships of the cardiac anatomy diagrammatically in two
dimensions. The relationships of the interatrial (IAS) and interventricular septa (IVS) are shown behind
the aorta and pulmonary artery and between the attachments of the tricuspid and mitral valves. Under-
standing of these important structures and relationships is essential to the understanding of the appli-
cation and use of lasers within the heart as described in the text.
FIGURE 9.3 Two-dimensional diagram of three-dimensional relationships of cardiac anatomy.
Figure 9.4 shows the passage of a catheter through the arterial tree from the brachial or femoral artery
as described in Figure 9.1, in the opposite direction of the arterial blood flow. The catheter can be passed
retrograde through the aortic valve into the left ventricular chamber to interrupt abnormal conduction
pathways high on the ventricular septum, as in position (b), or to drill channels through the ventricular
wall as in position (a). Thickened cardiac muscle can also be incised (cut) or excised (cut out, or cut
away) with a laser catheter (see Section 9.6.2).
A transvenous approach is shown in Figure9.5. The laser catheter is passed along the direction of the
flow of blood in the veins after needle puncture of a peripheral vein. The catheter enters the right side
of the heart from the superior or inferior vena cava. This approach is ideal for performing an atrial
septostomy ( position (b) ) or tricuspid or pulmonic valvulotomy (position (a) ), as well as to destroy
abnormal conduction fibers high on the right side of the ventricular septum, or, more commonly, along
their pathway from the superior vena caval–atrial junction to the interventricular septum (position (a)).
In addition to creating a sufficiently large atrial septostomy for blood to flow, the septostomy can be kept
small to allow passage of the catheter into the left side of the heart (position (c)). Pediatric cardiologists
have used such approaches for many years for diagnostic and some therapeutic procedures. (See text for
description and Sections 9.7.
9.4 Physics
The laser–tissue interactions that occur as laser energy is transferred into living tissue are complex; in
addition, the biologic reactions that result in each type of tissue are many and varied, and our under-
standing of them is, at best, rudimentary. Fortunately, the medical investigator can judge his results from
simple observations of patient complications, recurrences, and failures. However, to avoid needless and
possibly dangerous applications, the physician should have as thorough an understanding of as many
aspects as possible. The greater the understanding of basic science the physician has, and the greater the
understanding of the biologic response the physicist has, the more likely the success of an innovation.
FIGURE 9.4 Passage of a catheter through the arterial tree from the brachial or femoral artery in the opposite
direction of the arterial blood flow.
Such knowledge is also useful to the physician and basic scientist in understanding the marketing claims
of different equipment manufacturers. We describe these physical–chemical factors as they apply to the
cardiovascular system, particularly to laser angioplasty, and relate them to current knowledge of the
initiation of vessel wall injury and the hyperplastic biologic response that results in restenosis.
In general, the transfer of the light energy into a tissue depends on three factors:
1. The wavelength of the light
2. Its power density
3. The chemical and physical properties of the target
Figure9.613 shows the energy contained in photons of different wavelengths.
The differences in the absorption properties of the tissues of the blood vessel wall and the organic
portions of atherosclerotic plaque are very small, especially at the energy density levels needed to remove
tissue, although investigators have attempted to utilize such differences between normal vessel wall and
the atheroma to aim or direct the laser beam.14–20 However, for patients to have symptoms of atheroscle-
rotic disease, the lesions must be flow obstructing, and such lesions always contain inorganic calcium
phosphate particles dispersed throughout. Symptomatic, or “complex” atheromas are, therefore, mixed,
and contain both organic and inorganic portions. The inorganic portions contain less water than the
surrounding organic tissue. This is important because the energy–tissue interaction of such a sample is
far different from that of purely organic components. The absorption of the laser radiation by the tissue
is given by the equation:
I' ( intensity of light transmitted ) = I ( incident ) ( e -αL )

FIGURE 9.5 A transvenous approach.
FIGURE 9.6 Photon energy in electron-volts of some lasers currently in use or under investigation for medical
applications.
where “I” is the intensity of the light beam, “L” is the thickness of the tissue, and “α” is the absorptivity
of the target tissue. This relationship has an important consequence in the nature of the laser–tissue
interaction. If the absorption is strong, the photons penetrate to a depth of only 1/10th to 1/100th of a
mm from the tissue surface. Strong absorption tends to minimize the thermal damage to the underlying
areas because the diffusion of heat from the laser photons is minimized. Shortening the laser’s pulse also
minimizes the lateral spread of heat or thermal damage. The wavelengths of infrared photons correspond
in energy to vibrational and rotational sublevels of excitation of the molecules of all biological tissues.
This mode of energy transfer is known as “heat,” and is rapidly redistributed among the molecules
and raises the temperature of the target tissue. At power densities of a kilowatt/cm2 or more, especially
when the exposure time is in milliseconds (ms) or longer, the temperature of the exposed region can
rise to well above the boiling point of water. Here, the primary absorber of the photon becomes an
important consideration. Short-pulsed solid state lasers in the near-infrared such as the Er:YAG and the
Ho:YAG are well suited by the wavelength to be absorbed selectively by the water component in tissue.
The output of the CO2 laser is also in the infrared at a wavelength strongly absorbed by water, but the
lack of a suitable means to deliver this energy inside the blood vessel has limited its applications.
Because chemical changes that are brought about by ultraviolet photons are pervasive in our lives, a
field of study called photochemistry has evolved over the past hundred years. These chemical processes
include effects such as photosynthesis and sun tanning, as well as less desirable effects such as air pollution
(photochemical “smog”) and destruction of the ozone layer in the stratosphere. The absorption process
of ultraviolet lasers (mainly excimer lasers) is fundamentally different (Figure 9.7) from the absorption
process of the visible and IR wavelengths.
With UV, absorption leads to excitation of the electrons that hold the atoms of the structural molecules
together. This physical phenomenon is called electronic excitation. It conveys the energy of the photon
directly to the bonding electrons and facilitates the breakup of these bonds. The process of excitation
itself can cause the bond break, or it can happen subsequently in a few billionths of a second. The typical
bond of an organic molecule has energy of 3 to 4 electron volts. The energy of a photon at 193 nm (such
as ArF) is 6.2 eV. Although electronic excitation is due to a specific excitation process at a given wavelength,
the reactions that follow need not be equally specific. Heating effects as a result of UV excitation may
occur, due either to an inefficient bond-breaking process in which photons that fail to break a bond
merely heat the molecule, or the bond energy is considerably less than the energy of the photon so that
the excess is available for heating.
FIGURE 9.7 A schematic representation and comparison of the interaction of ultraviolet photons with tissue (top)
and with the interaction of infrared photons with tissue (bottom). The mesh represents filaments of structural protein
molecules interspersed with the water that constitutes the major part of the tissue. When the photons are of ultraviolet
wavelengths. Absorption is exclusively by the protein, which then undergoes bond-breaking and ablation. The water
is expelled mostly in a liquid state, along with the pieces of the protein and its gaseous products. When the photons
are of infrared wavelengths, it is the water that absorbs strongly to produce steam. The resulting explosion tears the
protein filaments apart.
The laser–tissue interaction is a collective process in which a stream of photons interacts with a slab
of tissue. The density of the photon stream, or power density, is one of two factors that control the density
of excited molecules in the tissue the instant the light beam strikes. At the power levels at which lasers
operate, the laser–tissue interaction is by no means limited to a given molecule’s being excited by just
one photon. Two, and even multi-photon excitations, become possible. Figure 9.8 is a plot of the matrix
of power densities and pulse widths currently employed in the gamut of laser–tissue interactions. This
is explained by: Fluence = Energy deposited/area,
where E is expressed in Joules (4.2 J = 1 Calorie) and
Power density = Energy deposited /time so that
Power density = Fluence /time.
The shorter the time, i.e., pulse width in which a given amount of energy, fluence, is delivered to a target,
the greater the likelihood that it will excite molecules by more than one photon.
Ablative photodecomposition and photodisruption strongly depend on such multi-photon processes.
The same amount of energy, if delivered in a longer pulse (toward the right in Figure 9.8) results in
simple single-photon reactions and heating effects.
The wavelength of the photon should always be kept in mind because the wavelength determines the
energy of the photon and therefore affects, and, in fact, may determine, the nature of the excitation
process. The mode of action for UV photons is specific and different from photons of longer wavelengths.
Only two types of interaction between the photon beam and the arterial wall (including the atheroma)
need be considered in the clinical application of coronary laser angioplasty. These are the interactions
brought about by a pulsed infrared laser whose photons are absorbed predominantly by the water in the
tissue, and by a pulsed ultraviolet laser whose photons are absorbed exclusively by the organic bodies.
Other modes of laser–tissue interaction that were tried and have largely been abandoned are: CW
argon and CW Nd:YAG, either alone through a fiber, or through a metal tipped fiber in which the beam
really did not interact with the tissue directly, but merely served to heat the metal tip in short periods
of time. This so called “hot-tip” is not a laser–pulse interaction. In a modification of the capping of these
fibers, the laser beam both heated the tip and partially escaped to interact with the tissue. Here again,
the output of the laser was in the visible spectrum, so that the interaction of the photons with the tissue
FIGURE 9.8 Power density and pulse width effects (Ready, 1978).21 A matrix showing how different laser–tissue
interactions relate to the power densities and pulse widths of the laser. Both scales are logarithmic. The diagonal
lines connect points of constant energy density (fluence) in joules/cm2.
was entirely thermal in nature. (An electrical source of heat energy that was reminiscent of a heating, or
soldering, iron was actually tried experimentally but was quickly abandoned due to excessive thermal
injury. Nonetheless, the thermal use of the laser continued for several years.)
The overall process of using the photon energy of a pulsed laser to cause a transformation in a tissue
can be sorted into three individual steps (Figure 9.9). These are: 1) absorption of the photon, 2) bond
breaking, and 3) ablation of the products. The last step does not always follow, as the first two steps can
leave a transformed material in place. The process of absorption comes first and is confined to the surface
layers of the tissue.
The term “ablation” refers to a specific miniature explosion that results from the extremely rapid
buildup of pressure from any cause, including photo-decomposition, which is non-thermal as well as
thermal. The term “ablative photodecomposition” refers specifically to the action of UV photons and is
not a thermal reaction. In contrast, the term “vaporization” refers specifically to a thermal process such
as occurs with non-UV laser reactions. It is exemplified by boiling water so that it turns into a vapor
stream. These terms are often used interchangeably by those inexperienced in the field, which has led to
confusion among cardiovascular physicians. Ophthalmologists and dermatologists are generally more
aware of the exact meaning of these terms and, in fact, the former group have found a near optimal
application of an excimer laser — to ablate essentially pure organic tissue. The 193 nm argon fluoride
excimer laser was recently approved by the FDA to correct myopia and astigmatism (PRK and PTK) by
ablating — not vaporizing — corneal tissue.
The pressure rise that occurs with ablation can momentarily be greater than 1000 atmospheres. When
ablation does occur, there is a shock wave that travels in a direction exactly opposite to the direction of
the expelled products: the shock wave travels into the tissue. Its presence has been detected and its passage
timed by a piezo-electric pressure sensor that can be attached to an isolated piece of tissue.22 The shock
wave shows the time scale in which ablation occurs. If the photons, especially in the ultraviolet, are
FIGURE 9.9 Transfer of photon energy into target issue. Hypothetical steps in the interaction of a laser beam with
tissue. Top: the laser radiation, which is defined by a mask, is absorbed; Middle: chemical bonds are broken in the
tissue by the photon energy; Bottom: the products ablate at supersonic velocities, leaving an etched sample.
delivered to the tissue in a sub-microsecond pulse, the shock wave also builds up in a similar scale of
time, demonstrating that the process of tissue breakup is quite rapid. An equally important point of
concern is the potential for damage to the artery wall that can be shock-induced. The shorter the laser
pulse, the higher the power density, (for a given energy content, see Figure 9.8) and the more violent the
force of ablation.
Because ablation and its shock wave are paired in magnitude by a physical law, the ablation that is
brought about by pulses of high power density cause shock damage that is readily detected. In biomedical
applications, the power densities should be kept as low as possible to achieve the desired result, but the
cumulative action of numerous ablative pulses must also be taken into account. Laser ablation of ather-
omas is achieved with a succession of laser light pulses delivered to the site by means of an optical fiber.
A single pulse removes (ablates) only a minute volume of the atheroma, the cross-section of this volume
being defined by the cross-section of the beam, and the depth being related to the fluence of the laser
pulse (Figure 9.10).
The normal arterial wall is also susceptible to these pulses, and its etch depth is also shown as a function
of fluence. A threshold fluence must always be exceeded before there is any ablation, and this threshold
varies with the absorption characteristics of the tissue.
Most, if not all, of these complex physical phenomena that occur with laser energy transfer had
been observed and were well known to scientists long before physicians began to apply lasers to
cardiovascular tissue. In addition to the obvious thermal effects and vaporization, scientists were aware
of many nonthermal effects such as recoil, pressure wave formation, ionization, free radical formation,
photochemically induced oxidation, and even multiphoton absorption processes and mechanically
induced shock waves.23–25
The development of the excimer laser in the mid 1970s, however, gave scientists a new tool capable
of heretofore unattainable ultraviolet wavelengths. Srinivasan was one of the first to study the effect of
these new excimer lasers. In 1982, he described chemical bond breaking and introduced the term
“ablative photodecomposition” to describe the interactive process between far ultraviolet radiation and
protein molecules, resulting in a photochemical breakdown of molecular bonds with virtually no
thermal effect.26,27
Excimer lasers with wavelengths typically between 151 and 351 nanometers (0.151 to 0.351 µm) contain
photons with 10 to 100 times more energy than any lasers previously available; these wavelengths
correspond to electronic and ionizing excitation.
As part of a study of the basic energy transfer process, Srinivasan’s group specifically studied the process
of ablation of vascular tissue by high-speed photography. The set-up is shown schematically in Figure 9.11
and the photo results in Figures 9.12–9.15.
FIGURE 9.10 Etch-curve for normal (continuous line) and atheromatous (dashed line) aortic wall at 249 nm and
351 nm. The energy density is the total value for a train of 250 pulses.
FIGURE 9.11 Schematic diagram of experiment.
A sample of artery was obtained at postmortem, and laser energy at 308 nm was channeled through
a quartz fiber of 600 µm cross-section to ablate the inner surface of the artery in air. The laser pulse had
a half-width of 30 ns. To “freeze” the motion of the ablated products at any time t following the ablation
pulse, a dye laser that produced visible light pulses of less than 1 ns duration was used to provide
illumination for the photo. A timer (trigger) connected to both lasers allowed the dye laser (which
provided the “flash” illumination for flash photography) to be fired at a controlled delay after the
ultraviolet laser pulse had been fired.
The purpose of these photos was to investigate the ablation process itself when the optical fiber was
placed well above the tissue surface (Figure 9.12) and when the fiber was in contact with the target tissue
(Figure 9.14). Ablation of the normal wall and atheroma was also compared. Many studies by other
investigators have since confirmed Srinivasan’s work and advanced our understanding of this ablation
process and the consequences of the blast wave that is propagated.
FIGURE 9.12 Ablation of intimal wall by a single 308 nm pulse seen in profile. The vertical cylinder in the middle
of the frame is the distal end of a 600 µm thick optical fiber stripped of its outer protective plastic coating. The frame
covers approximately 3 mm by 3 mm. The delay between the excimer pulse and the dye laser pulse: 750 ns.
FIGURE 9.13 Ablation of soft plaque by a single 308 µm pulse seen in profile. The delay time is 1µs.
FIGURE 9.14 Ablation of intimal wall by a single 308 µm pulse seen in profile. The distal end of the fiber is just in
contact with the surface of the tissue. The delay time is 1000 ns.
FIGURE 9.15 Ablation of soft plaque, predominantly organic tissue with little or no inorganic crystals, by a single 308
µm pulse seen in profile. The distal end of the fiber is just in contact with the surface of the tissue. The delay time is 30 µs.
Because ablation is an explosion brought about by the generation of an enormous pressure inside the
tissue, the products emerge from the surface at high speed, which can exceed the velocity of sound in
air. When this stream meets the air at atmospheric pressure, a blast wave is created that becomes visible
as a gas bubble because of the great difference in the refractive indices. This blast wave is usually followed
within microseconds by a stream of debris referred to as a “plume,” which consists of solid particles of
tissue or atheroma and water droplets that are driven by the gas stream. Both the blast wave and the
plume slow down as they expand and cool down.
The blast wave is seen to emerge from the surface of normal artery in less than 750 ns in Figure 9.13.
and expand progressively. Its average velocity is 7.5 × 105 cm/second, which is in the supersonic range.
The profile of the blast wave is complex and actually shows an inverted pattern. It is possible that the
fiber end, which is only 0.5 mm from the tissue surface, causes a reflection of the blast wave. The
velocity for the front edge of the wave is 4 × 104 cm/second, slightly greater than the velocity of sound
in air. In Figure 9.14, the ablation of soft plaque is seen to be much different from the ablation of the
artery wall. The blast wave is only faintly seen. The plume emerges with a velocity similar to that noted
in the artery wall, but its density (opacity) is far greater, which suggests that the amount of solid and
liquid material ablated is also greater. Material is also ejected from the surface for a much longer time
— up to 15 µs.
The effect of contact between the fiber end and the intimal wall on the ablation is pictured in
Figure 9.14. As the blast wave is propelled by rapidly expanding gases at high pressure, it manages
to escape from the confinement of the fiber end. In polymer ablation, it has been estimated that
the gas pressure built up during ablation might be of the order of 100 to 1000 atmospheres. It is
not surprising that gases at such high pressure would escape in the interstices between the fiber and
the tissue surface. However, the plume is almost totally suppressed, with just a trace of debris being
visible.
Figure 9.15 shows that, when the fiber tip is gently pressed against the surface of the soft plaque, the
result is different from that seen when the tip is barely in contact with the intimal wall. There is a violent
upheaval of the surface around the fiber tip as the confined gases burst out of the limited volume in
which they are formed.
During laser angioplasty, the fiber tip is steadily advanced into the plaque to open a channel. A series
of high-speed photos were taken at a constant time delay of 60 sec between the ultraviolet pulse and the
“flash” pulse to determine how the progress of the tip into the plaque affects the ablation caused by
successive pulses. The first pulse was fired with the fiber tip pressed gently to the surface of the plaque.
Violent bursting of the surface is observed. A second pulse was delivered with the fiber tip not advanced.
A similar outburst was not seen. However, when a pulse was fired after the tip was advanced until contact
with the tissue surface was again felt, and with the fiber end is buried inside the plaque, the ablation is
once again as violent as with the first pulse.
This series of photographs reveals an additional important problem in the performance of laser
angioplasty. The interventionalist positions the distal end of the laser catheter against the obstruction
and uses the tactile response from firm contact between the fiber end and the tissue surface as guidance.
As the catheter moves farther into the plaque, a tight channel is formed. Kar and Biamino28 also noted
this phenomenon. The enclosure of the distal end of the catheter into the plaque traps the expanding
gases until only a violent ablation can permit their escape. This can cause an unacceptable stress to the
walls of the vascular system.
Although these photographs were obtained using a UV laser, similar explosive reactions occur with
all lasers. The pressure buildup preceding ablation may be due to different laser–tissue reactions, but the
effects of the ablation depend only on the explosive power behind the phenomenon.
The effect of an excimer laser on the inorganic molecules of calcium phosphate that are mixed
throughout the organic stroma of plaque appears to be mechanical (See Figure 9.16). The inorganic
crystals appear to be pulverized (see Figure 9.17). Several mechanisms and processes involved in
pulverizing the calcium include:29
FIGURE 9.16 Effect of excimer laser on pure organic portion of human atherosclerotic plaque. Because of explosive
mechanical processes, even pure organic portions are ripped and torn.
1. Although there is far less water in calcium phosphate than in the organic part of the plaque, the
water that is present is disrupted by secondary and possibly thermal means to produce steam.
This steam explosion can shatter the adjacent crystalline material.
2. Absorption of laser energy by the remaining dehydrated inorganic mineral crystals can result in
differential byproducts expansion, which, in turn, can cause shattering and disruption.
3. Photochemical disruption of the chemical bonds that bind the inorganic calcium phosphate
molecules together is now believed to occur.
4. Mechanical vibrational disruption of inorganic molecules has long been known to occur in non-
biologic systems.
5. Energy that has been stored within the inorganic plaque as it was formed is released. This includes
additional mechanical and possibly some thermal energy.
The potential medical application of “ablative photodecomposition,” first described by Srinivasan in
1982 and summarized by him in 1986,30 1989,31 and 1994,32 was very quickly recognized as a possible
means of avoiding thermal damage and efforts were directed to develop a fiberoptic delivery system. To
transmit the very high peak powers of these very short pulses, the pulse duration was stretched. While
threshold for ablative photodecomposition may no longer be achieved, the energy can be delivered
through the fiber in very short pulses that are still measured in nanoseconds. Absorption depth is very
shallow so that thermal damage has not been noted. Mechanical shock wave damage, however, is now
believed to be responsible for the high incidence of restenosis.
FIGURE 9.17 Effect of 308 excimer laser on section of “hard” or calcified plaque. Note the fragmentation into smaller
particles but the absence of evidence of melting of these fragments, consistent with mechanical energy transfer.
9.5 Angioplasty (with Mark H. Wholey, M.D.)

The overwhelming cause of incapacitating occlusive vascular disease is the metabolic disorder termed
atherosclerosis. (The older term, arteriosclerosis, means literally just hardening of the arteries). The
normal arterial structure is depicted graphically in Figure 9.18. The nonsurgical endovascular treatment
of occlusive arterial disease including the use of lasers is generally acknowledged to have begun with
Andreas Gruentzig’s report in 197933 that he had successfully opened both coronary and peripheral
arteries by inflating a balloon attached to a thin flexible catheter. Charles Dotter34 had reported as early
as 1964 on a method of progressive dilation of peripheral arteries with flexible but uncompressible rods
or catheters. Although Dotter’s report provided the stimulus for Gruentzig, it generated more controversy
than enthusiasm with other medical investigators. Nonetheless, it is now recognized that “soft” plaque
is often pushed aside by stiff catheters. This has been termed “Dottering” and may occur with any rigid
device including “hot tips” and laser catheters. The balloon dilation method of Gruentzig however,
combined with the Seldinger technique, provided a simplified means of opening vessels of any size by
using only a needle puncture followed by the insertion of a single narrow catheter. Many other mechanical
devices have been developed since, but balloon dilation remains the standard nonsurgical technique.
As a result of the success of percutaneous transluminal peripheral and coronary angioplasty
(PTA/PTCA), experiments with the commercially available lasers at that time, the CW Nd:YAG, argon
and CO2 were done on excised human arteries or on cholesterol plaques that had been induced by
diet in experimental animals.35–38 Dead tissue does not react and animal tissue bears no true resem-
blance to human atherosclerosis. All of these lasers “removed” atherosclerotic material but the human
endovascular reaction, however, could not be assessed or even appreciated at that time. Despite the
lack of scientific background, human experiments were begun in 1983 with CW argon lasers coupled
to bare silica fibers.39,40
Occluded peripheral and coronary arteries were opened, proving the feasibility of the method, but
the perforation rate was unacceptably high,41 and occlusion usually recurred within several months.
b
a
FIGURE 9.18 Schematic of a normal artery cross-section. (a) intima, inner-lining layer of 1-3 cells thickness. (b)
internal elastic lamella; this is a tissue plane within the media that demarcates the subjacent intima from the media;
it provides a natural surgical cleavage plane that enables the surgeon to peel away the plaque, a procedure called
endarterectomy. (c) media. (d) external elastic lamella, the tissue plane that separates the media from the exterior
adventitia. (e) outermost rim of media subjacent to the most outer layer, (f) adventitia, the most outer layer of the
vessel wall.
The perforation rate was decreased by capping the tip of the fiber with metal ,but now there was no
true laser–tissue interaction because the laser merely served to heat this “hot tip.”42
Nonetheless, with the encouragement of marketing and sales claims, some physicians actually per-
formed the equivalent of running hot pokers through the vascular system to cook fatty droplets. These
initial clinical experiments were reported enthusiastically in the medical literature and lay press and were
vigorously promoted by the marketing and advertising efforts of some newly formed entrepreneurial
companies. This period has been described as “… overly aggressive and premature clinical use of poorly
characterized laser angioplasty systems … fueled by exaggerated expectations … and a limited under-
standing of laser microsurgery.”43
Rutherford summarized deviations from what had been traditional and acceptable standards of ethical
medical practice,44 noting that they included, “Problematic reporting practices … .elimination of initial
failures … not distinguishing between primary and secondary patency … counting re-treated patients
as successes … using subjective rather than objective criteria … comparison with historical controls
selected with self-serving bias … (and) failure to follow uniform standards in evaluating and reporting
results ….” He concluded that, “Clearly some practical constraints must be imposed to curb this tendency
toward unreasonable commercialism.” Unfortunately, this was the atmosphere into which laser angio-
plasty was born. Zarins45 has also reviewed the lack of constraints during this period, especially in regard
to conflicts within the medical specialties, and characterized the issues raised between the various medical
specialties as “The Vascular Wars of 1988.”
Enthusiasm fueled by the unrealistic optimism of marketing hype reached the point that some patients
actually demanded laser angioplasty and searched for physicians and hospitals with any type of a laser.
Rutherford46 reviewed the literature at this time and estimated that, despite a success rate of less than
50% in lesions longer than 7 cm (which is a reasonable assessment of the majority of clinical problems
and extent of the disease) in less than 2 years, more than 20,000 hot-tip angioplasties were performed
in more than 300 “laser centers” in the United States alone. In addition to the many adverse effects of
the hot tip, more problems are not so obvious. Iodine has been shown to be liberated from the contrast
media during the procedure, both in the form of soluble free iodine and in solid elemental iodine debris
that can break off and embolize. Free iodine embolizes immediately. In addition, both blood and contrast
agents have been shown to boil in proximity to the probe tip. Blood so heated exhibits markedly
accelerated clotting properties.47
The initial accomplishment of pushing metal-capped laser-heated fiber beyond an obstruction now
seems more probably the result of technical advances in guidewire and catheter technology plus the effect
of “Dottering,” rather than the result of any laser phenomenon.
Surprisingly, however, FDA approval was quickly granted for peripheral use, mainly on the basis of the
distorted claims of acute, or immediate, “technical” success. Gradually, as long-term results were reported,
it became apparent that there was no significant improvement in long-term patency, and that restenosis
quickly occurred. Results with some subsets of patients were not only worse than with either surgery or
PTA, but worse than the natural history of the disease untreated.48
Today, the hot-tip has essentially been abandoned as a peripheral vascular treatment in favor of PTA or
one of the other FDA-approved mechanical devices, or for surgical bypass. Attempts to use these thermal
systems within the coronary arteries were quickly abandoned, as the heart very rapidly demonstrated that
it cannot tolerate the acute thermal insult.49–52
Srinivasan had almost immediately recognized the potential medical application of the non-thermal
ablative photodecomposition of excimer lasers that he had described.53,54
In a review of some of the commercial development of one of the excimer lasers for angioplasty, Forrester
et. al.55 stated, “As late entrants into this field we found that there was a major limitation for vascular
application: the thermal energy … frequently caused vascular spasm and thrombosis … disappointed by
our in-vitro results, we decided to review the materials literature … to our amazement we found that the
excimer laser … produced cuts through inorganic material without thermal damage.”
Although Forrester et. al. credit Srinivasan and Leigh as the source of this information, they cite the date
of their publication incorrectly as 1984 (it was 1982) and misidentify the pure organic target material that
these IBM researchers used as the basis for their initial ablation experiments (see Reference # 27). The
correct information is obviously of great importance, not only to establish proper priority, but to understand
the evolution of the research in this field, the rationale behind it, and, ultimately, to understand the science.
Atherosclerotic tissue contains a mixture of inorganic salts in an organic matrix. The transfer of the excimer’s
high-intensity energy into this mixed tissue is complex and has been shown to result in ripping and tearing
of adjacent tissue. This mechanical effect is discussed in Section 9.4.
Forrester et. al. go on, “… we packed some tissue in a suitcase and flew to the Argonne National
Laboratories in Illinois to use their excimer laser on our tissue. The result was spectacular ….” The basic
scientists at the Argonne National Labs were not only aware of the IBM scientists’ observations and this
new theory of energy transfer that they had termed “ablative photodecomposition,” but the Argonne
scientists understood the significance of these observations. The Argonne scientists carried out the initial
experiments with the 308 nm excimer (xenon chloride) for laser angioplasty and received the patent56 for
this achievement. Forrester’s group at Cedars-Sinai Hospital in Los Angeles then collaborated with scientists
from the nearby Jet Propulsion Labs (JPL) in Pasadena to form the Advanced Interventional Systems
Company (AIS) for the purpose of developing and commercializing a laser angioplasty system. Lauden-
slager,57 a former JPL scientist who became the chief scientist at AIS, has acknowledged that, “… it was
Srinivasan’s work with excimer lasers that was the experimental model for all of our work on laser–tissue
interactions.” AIS has since merged with the Spectranetics Company.
Fiber optic delivery systems were developed to transmit the very high peak powers of the micro pulses
of excimer lasers.58,59 Technical improvements with guide wires, catheters, and control systems including
fiber optic visualization (angioscopy) and intraluminal ultrasound, resulted in a very high rate of immediate
or technical success. Unfortunately, patency was not maintained and restenosis rates were again disappoint-
ingly high. In addition, the laser catheters are unable to create sufficiently large lumens, meaning that
PTA/PTCA injury is still required for most patients. This dilemma of superimposing a second PTA/PTCA
injury on the laser injury to achieve an adequate lumen has not been solved. A report60 that restenosis
seemed to be lower for laser treatment alone than for laser plus PTCA has not been confirmed and, in fact,
the reverse has also been claimed..61
Nonetheless, in October 1990, the Medical Advisory Panel to the FDA recommended approval of the
AIS Company’s excimer laser system with a 1.6 mm diameter catheter for ostial lesions and diffuse
coronary artery obstructions greater than 20 mm in length. Clinicians have continued to try to identify
subsets of disease patterns for which this laser system might offer some improvement (efficacy) over the
poor results achieved with PTCA alone,62–65 including heavily calcified long66or ostial lesions,67 failed
PTA/PTCA, vein grafts,68 total occlusions,69 and thrombosis. Experience with the AIS system through
August,1990 at 15 institutions on 1,519 lesions in 1284 patients was reviewed by Margolis and Mehta.70
Experience with this laser system in 3000 patients from 33 laser centers was reported by Litvack, et al.
in 1994. While total numbers were greater, results were essentially the same as in the earlier reports.
In November 1991, the FDA Medical Advisory Panel recommended approval of the Spectranetics
Company’s excimer laser system with 1.4- and l.7-mm-diameter catheters for the following subsets of
coronary obstructions:
1. Diffuse and greater than 10 mm in length
2. Aorto-ostial
3. Calcified
4. Total
5. PTCA failures
6. Saphenous vein bypass grafts
The clinical studies on which this decision was based have been published.71,72
Haase et al. summarized results with the Technolas Excimer Laser system.73 This German system has
not been presented to the FDA for U.S. approval. The company has modified its original system in an
attempt to decrease the accompanying mechanical shock, or blast, wave.74 This group acknowledged that
the results of excimer laser angioplasty ablation did not result in an improved patency rate due to the
high incidence of restenosis. They attributed this to pressure-wave generation. The catheter was modified
by Technolas physicists and engineers to contain eight sections of 20 fibers each, for a total of 160 fibers
of 50 microns each. With the use of a scanner, the individual sections were fired one at a time, thus
reducing the amount of energy delivered with each pulse. Mean fluence was 49+/-2 mJ/mm2. This
effectively reduced the shock wave in bench tests. Delivering laser pulses into a glass of water, the pulse
wave of the ordinary delivery system feels like a hammer blow, while the pulse wave from the SELCA
catheter is barely detectable.75 Animal experiments also showed that mechanical acoustical injury and
the proliferative response were both reduced, while ablation was maintained.76
This system was then used in 28 patients with a 1.5mm catheter and four patients with a 1.8mm
catheter. The mean reduction in the coronary artery stenosis all 32 patients was from pretreatment of
85+/-10% to postlaser treatment of 57+/-20%. However, half of the patients required additional imme-
diate balloon PTCA, five of these being necessary because of abrupt closure after the laser treatment.
Dissection was observed in 10 patients. Restenosis, however, was noted in 14 of the 24 patients who
agreed to angiography within 6 months posttreatment. There was no difference in subgroup analysis
between those patients who had laser only and those with laser plus balloon dilation. These authors point
out the importance of delivering the energy axially by catheters of the proper size. They conclude that
larger-diameter catheters need to be developed to properly evaluate their new system.
Despite the failure of any type of laser to achieve the hoped-for improvement in patency over
PTA/PTCA, clinical success has been claimed in selected subsets of patients77 who have been able to delay
surgical bypass (see Figures 9.19 and 9.20). The arrows point to areas of atherosclerotic occlusive disease.
Note the beneficial effect of the laser treatment.
Efforts continue to salvage the excimer laser and to find some subset of patients for whom it might
offer an advantage. At the same time, it has become apparent that not only is the “POBA” (plain old
balloon angioplasty) adequate for the vast majority of lesions, but that there are some subsets of lesions
for which PTCA is superior to the laser. In addition, Appleman et al.78 showed that the excimer yields
an adverse clinical outcome of tandem lesions.
Spectranetics is also sponsoring clinical studies using their device to help remove pacemaker electrode
leads that have become fibrosed within the right ventricle. This use of a laser catheter was first suggested
by Rao and Flaherty,79 who also demonstrated its feasibility in animal experiments. This may be an
appropriate use for the excimer because the electrode is trapped within essentially pure fibrous tissue;
FIGURE 9.19 Pre-angioplasty. Arrows point to areas of atherosclerotic occlusive disease.
FIGURE 9.20 Post-angioplasty. Note the beneficial effects of laser treatment.
there is no inorganic calcium salt to interfere with the laser’s mode of ablation of tissue. The company
is also preparing a separate clinical study using its excimer laser to reopen thrombosed stents (Figure
9.23). This, too, could be an ideal application of the technology, because the occlusion of stents within
vessels is an urgent medical problem and is usually recognized quickly; these obstructions are almost
always either thrombus or early hyperplasia, and neither contain inorganic calcium.
FIGURE 9.21 Arterial and venous vessels within the muscular layers of the ventricular wall, and the interrelationship
of the arterioles, venules and capillary vascular bed to the ventricular wall.
FIGURE 9.22 Vineberg Procedure. The internal mammary artery is dissected free and divided and then pulled into
a tunnel made into the ischemic wall of the ventricle. The side branches are not ligated, but are allowed to bleed
freely to communicate with the intramural vessels.
The totally occluded vessel remains a therapeutic problem that usually requires surgical bypass.
Although a guidewire can often be passed through short occlusions, it is not often possible to introduce
a dilating balloon or device. In an effort to take advantage of the interventionalists’ ability to pass a
guidewire through such occlusions, the Specranetics Company has designed a laser tip guidewire. Reports
of the initial clinical studies with this device indicate that it could be passed in 58% to 63% of lesions.
The perforation rate was 21% to 27%, and all patients required additional endoluminal treatment, either
by laser or balloon angioplasty, artherectomy or stent replacement. “Treatment success” in a subset of 56
patients was claimed to be 61%.80
The term “laser-assisted balloon angioplasty” requires special mention. It has been used by some
authors to refer to the fact that balloon angioplasty is usually needed after laser angioplasty, but it had
a very specific meaning in a series of clinical experiments when a laser was used to deliver thermal energy,
or heat, to smooth and stiffen the ruptured walls of vessels following the balloon angioplasty. This latter
application is more related to tissue welding than angioplasty. The concept was suggested by Spears,81
who noted that CW Nd:YAG energy could fuse or weld the fissures and cracks that result as part of the
necessary plaque disruption with PTCA. From this observation, Spears developed the concept that
delivery of controlled laser energy, essentially as heat, to the tissue surrounding the PTCA area, could
modify or chemically alter the collagen to become a biologic stent. Clinical trials confirmed that the
tissue surrounding the PTCA could be modified by the heat, but unfortunately, the almost infinite
variability of the composition and thickness of the tissue receiving the heat resulted in an unacceptable
incidence of occlusions81 and the procedure has been abandoned.82
9.6 Transmyocardial Laser Revascularization (TMLR)

In 1977, Mirhoseini83 reported using a CO2 laser to create channels through the ventricular wall into the
left ventricular cavity of dogs to nourish ischemic myocardium. He showed patent, endothelialized
connections between the ventricular cavity and intramural arterioles 2 1/2 months postop. Although this
procedure has an anatomic and physiologic basis, there is considerable confusion, controversy and even
skepticism about its potential.
Direct coronary artery connections through intramyocardial sinusoids and arterioles to the intracar-
diac blood were first described by Vieussens (1705) and Thebesius (1708). These connections have
intrigued surgeons and cardiologists at least since 1898, when Pratt kept a cat’s heart beating by perfusing
blood into the ventricle. In 1933, Wearn et al.84 identified two types of connections, arterio-sinusoidal
and arterio-luminal, and pointed out that, because they communicate between the cardiac chambers and
the coronary vessels, they might serve as a source of blood for ischemic myocardium. These vessels, long
before open-heart surgery was possible, quickly became the basis for many attempts at revascularization,
most prominently Vineberg’s implantation of an open, bleeding internal mammary artery directly,85–87
and Beck’s arterialization of the coronary sinus88,89 and coronary sinus retroperfusion operations.90,91
These procedures were done on beating hearts long before the advent of heart-lung bypass or selective
angiography. Vineberg summarized his personal experience over more than 30 years, including post op
angiography and proposed explanations for why others had not achieved comparable results.92 Shrager
recently reviewed the “… remarkably hard fought debate that raged over the value of IMA implantation,”
but “… recent reports of long-term graft patency and clear patient benefit … reinforce the “belief that
Vineberg should be given more credit … and that IMA implantation should not be relegated to the status
of historical curiosity.” Between 1946 and 1975, more than 10,000 of these procedures had been per-
formed, but follow-up data was contradictory and “… this raging debate was rendered moot, however,
by the advent of coronary bypass grafting. … the debate over the Vineberg procedure was never resolved,
only abandoned. Once bypass was possible, the medical community lost interest in implantation.”93
Likewise, the Beck operations were done on thousands of patients in this era. Bailey94 reported his
own series of 53 patients with vein segments anastomosed between the aorta and the coronary sinus plus
18 more direct anastomoses, all done on a beating heart before the era of heart-lung bypass. In his
encyclopedic review of cardiac surgery, Shumacker95 recounts additional efforts to utilize these intramy-
ocardial anastomoses by arterializing the coronary sinus in patients.
These surgeries were undertaken without the benefit of coronary angiography, which had not yet been
developed. It seems clear now that the major problem was the inability to identify the extent of disease
and therefore to make the proper patient selection. Also, it was ultimately shown that it takes several
weeks for the new vessels to be able to carry significant amounts of blood. Many patients died during
this period, although those long-term survivors who eventually had selective angiograms demonstrated
functioning anastomoses and the value of the method for the appropriate patient.
In 1956, Goldman et al.96 suggested a method of using the intracavitary blood to revascularize the
heart directly. They cut multiple holes in a segment of artery or plastic tubing that was passed through
the ventricular wall into the ventricular cavity and then back out through the wall. Although animal
results were encouraging, there is no record that this ingenious procedure was ever performed in patients.
In 1965, Sen pointed out that the reptilian heart derives its blood entirely from the ventricular cavities
through many small channels into the sinusoidal-capillary bed.97 Following extensive animal experimental
studies, he performed multiple transmyocardial needle punctures in four patients with acute infarction, but
none survived. He noted that other surgeons had tried the procedure as a last resort for acute infarction —
also without success.98 A year later, Hershey and White did report a survivor who was successfully resuscitated
by transmyocardial needle punctures after having been refractory to all other forms of treatment.99,100
However, in 1969, Pifarre et al.101 claimed that, even if the anastomoses remained patent, it was
impossible for an adequate amount of blood to perfuse the ventricular mass because of the heart’s systolic
contraction. This, coupled with the development of the bypass operation, brought transmyocardial
revascularization efforts to a halt until Mirhoseini’s work. Some surgeons, however, continued to use
retrograde coronary sinus perfusion.102–105 Studies of retrograde perfusion have shown that, under certain
conditions, anastomotic collateral flow does supply at least a part of the heart muscle.106–109
Many patients have such diffuse disease that they are not candidates for either coronary artery
bypass surgery or balloon dilation and do not respond well to drugs. They suffer from recurrent angina
and congestive heart failure and face early demise. Cardiac transplantation may help some of these
patients; however, the 2200 donor hearts that become available each year110 are inadequate to meet
the needs of potential recipients. The lack of adequate treatment for these patients stimulated research
into alternative means for revascularizing ischemic myocardium and renewed interest in some of the
previously abandoned methods. Mirhoseini111–115 created 20 to 30 CO2 laser channels in ischemic
myocardium of dogs and demonstrated significantly increased survival in those treated (83%) com-
pared with controls (0%). Patency after 6 months was also observed, suggesting that laser channels
eventually reendothelialize. Patency of laser channels 5 years post-op has been reported by ventricu-
lography and histology.116–119
In 1986, Mirhoseini120 reported the first clinical use of this technique in a patient who underwent
quadruple coronary by-pass surgery and supplemental transmyocardial laser revascularization to an
ischemic area that could not be bypassed. Neither arrhythmias nor significant cardiac enzyme increase
were reported. Although this result was encouraging, Mirhoseini did not study myocardial contractility
or myocardial perfusion. Solid evidence of feasibility and safety was therefore lacking, and the medical
community remained unconvinced of the efficacy of laser myocardial revascularization.
A 2.4 micron Thulium-Holmium-Chromium:YAG (THC:YAG) laser decreased the infarct size area
fourfold when compared with untreated animals.121 Because this laser can be conducted by optic fibers,
an endocardial approach was chosen. A guiding catheter was introduced into the left ventricle across the
mitral valve and the laser was applied from the endocardial surface to create nontransmural channels.
The versatility of this laser and the potential utilization of optic fibers in a closed-chest procedure
encouraged pursuit of further studies.
This group then studied the effect of the THC:YAG laser on regional myocardial performance using
regional preload recruitable stroke work (RPRSW), which is a load-insensitive index of contractil-
ity.122,123 A group of dogs was divided into two sets, one of controls and one of laser-treated animals.
Left ventricular pressure was measured with micro-manometers. Myocardial segment length was
measured with micrometry transducers and cardiac output was measured by thermo-dilution. The
area at risk was identified prior to performing the laser channels by brief occlusion of the left anterior
descending coronary artery. Intramural laser channels were then made with a fiber 600 micron in
diameter from the endocardial surface with parameters as follows: 600 mW,10 Hertz, and 20 repetitions,
totaling 12 joules/channel and averaging 3 channels/ cm with the total of channels per animal being
approximately 40.
The left main anterior descending artery was ligated for 90 minutes and then reperfused for 6 hours.
Pre-load was varied by temporary inflow occlusion. Although the area at risk in control animals became
dyskinetic, in laser-treated animals, RPRSW remained positive and contractility was preserved. Myocar-
dial dyskenisis was prevented and contractility was preserved. Microscopic exam demonstrated patent
channels but with collateral thermal damage.
Subsequently, experiments124 using an acute infarct model showed that contractility was improved
in the ischemic region when compared with controls. These studies demonstrate that small conduits
from the ventricular cavity can preserve regional myocardial contractility, indirectly implying that
myocardial perfusion was present.
However, there are also negative reports with this procedure.125 Whittaker et al.126 used Ho:YAG to
make l mm diameter channels from the epicardial surface of dog hearts into cyanotic myocardium after
ligating the coronary artery, similar to Sen’s attempts to treat patients with acute infarcts. With an 80
watt CO2 laser, Landreneau et al. created l mm diameter channels every 3 to 5 mm in the area supplied
by the left anterior descending coronary artery. They found no benefit following acute ischemia.127
The numerous differences among experimental models not only makes comparison difficult, there is
even controversy over what the microscopic sections reveal.128
Clinical trials with a specially made CO2 laser are currently under way at several centers. Encour-
aging results have been reported using computer-controlled 35- to 60-millisecond pulses of an 850
watt CO2 laser to bore 15 to 30 1 millimeter diameter transmural channels. Anginal class has been
reduced significantly with some preliminary evidence by nuclear radiography of improved perfusion
of previously ischemic areas.129,130 The technique can actually be used without the heart-lung machine
(placing the patient on cardiopulmonary bypass), another advantage for these very sick patients.131
Specific subsets of those patients who might benefit and those who would probably not are being
identified.132
In Germany, the Laser-Medicine-Zentrum Research Institute and the Technolas Company have col-
laborated with surgeons in Berlin with an operative approach using an excimer laser. Results are also
encouraging.133
The mechanism of the improved perfusion is thought to be due to the created channels, however,
alternative hypotheses such as stimulated angiogenesis from the laser treatment have also been sug-
gested.134 The average hospital stay has been only 9 days with an operative mortality of approximately
10%. None of the deaths appear to be secondary to the laser intervention.
A carbon dioxide laser was also used to create transmyocardial channels in five canine hearts, using
800W, 40J/pulse, and an average of 12 channels per heart, or approximately 1 channel per 2 cm2. Hearts
were then arrested and excised, the aortic valve was closed to prevent any LV ejection, the main coronaries
were cannulated, and perfusion was established from a second dog heart. A cannula was inserted into
the LV to control LV filling and afterload pressure. The LAD and epicardial vessels were ligated to make
the channel region ischemic. Colored microspheres were injected into the perfusate and the number of
spheres per gram of myocardium was measured. Blood from within the LV did not perfuse the myocar-
dium through the TML channels.135,136
Whittaker and Kloner used a 600 micron diameter fiber and pulse energy of 9mJ at 20Hz to make
three TML channels in rat hearts. These were the parameters that they had found made the widest
channels with the least thermal damage in vitro. After allowing for healing, these hearts were challenged
with 90 min of coronary artery occlusion. The authors measured the amount of necrosis and counted
the open channels. They found that open channels had less fibrosis surrounding them than did the closed
channels. These investigators proposed that the less thermal injury as manifested by fibrosis, the better
the chances for the patency of the channels.137,138
De Guzman et al.139,140 attached the CO2 laser output beam to a thorascope and were able to create
TMLR channels in five living pigs. Like the transvenous catheter approach, this technique avoids the
need for an incision into the chest (thoracotomy). The thoracoscope is introduced into the pericardial
cavity through a small intercostal incision.
Additional follow-up is necessary to support the initial encouraging results. Future research projects
should be designed to search for direct evidence of myocardial perfusion by the laser channels. Important
issues, such as the relationship between channel morphology, the radius of perfusion from each channel,
and the timing of perfusion through channels in relation to the cardiac cycle should be better understood.
The optimal wavelength is not yet known; there are theoretical advantages to an IR laser in terms of
short penetration depth and highly selective absorption, yet shock wave damage could be critical to
patients with already badly damaged hearts. The UV offers the possibility of less thermal and less shock
wave injury. If the technique can be shown to be of value, a transvenous catheter system would be much
preferred to an operative open-chest approach. All of these factors remain to be worked out. Nevertheless,
this technology remains one of the most promising, albeit challenging, applications of lasers in the
cardiovascular system.

(with Osvaldo J. Yano, M.D. and Mehmet C. Oz, M.D.)
The attention given to laser angioplasty has almost obscured other potential cardiovascular applications
where relatively homogeneous tissue may actually afford a better opportunity for success. Lasers are being
used either at surgery, or by an endocavitary catheter to create transmyocardial channels to increase
blood flow to ischemic myocardium (TMLC), to ablate arrhythmogenic foci, for valvulotomy, valvulo-
plasty, septostomy, myotomy, myomectomy, welding and endarterectomy. The cardiovascular structures
are shown elsewhere and described in the appropriate text.
9.7.1 Valvulotomy and Debridement

Congenital narrowing of the cardiac valves can be treated by simple incision along the fused cusps. The
use of a laser catheter to open these valves was suggested by Macruz et al. in 1981,141 Riemenschneider et
al.142 and Gessman et al.143,144 in 1983 and 1984. Rao145,146 has recently reviewed transcatheter methods of
relieving pulmonary stenosis, including the use of the laser. This treatment is still experimental, but appears
to be a promising alternative to surgery in neonates with these specific conditions; success continues to
be reported sporadically. Since1991, pediatric cardiologists at Leeds, UK have used a laser to open a
congenitally closed pulmonic valve in nine infants aged 1 to 70 days weighing 2.1 to 4.7 kgm. The distal
pulmonary artery trunk beyond the imperforate valve was shown to be present. The laser catheter was
manipulated into the right ventricle, below the imperforate pulmonic valve with a 0.018 in guide wire.
One to three bursts of CW laser energy of 3 to 5 Watts lasting 3 to 5 seconds allowed passage of the guide
wire beyond the valve. This was followed by passage of a coronary artery dilation balloon to dilate the PV.147
Long-standing mitral and aortic stenosis and insufficiency are far more distorted and repair is much
more difficult. Until satisfactory valve prostheses became available, valvulotomy and various plastic pro-
cedures, including debridement (the manual peeling away and excision of fibrous and calcified tissue from
the underlying valve tissue), were the only means of correction.148–152 Results were generally poor due to
scarring, even when the procedure was done perfectly. With the development of reliable valve prostheses
in the early 1960s, these unreliable and time consuming procedures are performed far less often.
However, lasers have been proposed as a means of achieving more precise debridement with less
damage to the underlying supporting tissue. But studies of excised valves, however, showed both the CO2
and argon lasers to be ineffective.153 Using a variety of near IR lasers in the range of 2 to 3 µm wavelengths,
Nuss, et.al.154 showed these lasers to be effective in ablating bone with minimal thermal or shock wave
damage. The absorption peaks of calcium phosphate and hydroxyapatite (the chemical structure of bone
as well as the calcium deposits in atherosclerotic plaque)155–159 had long been known to be at 3.1 µm and
2.94 µm respectively.160 This knowledge is the basis for testing the near IR lasers on calcified atherosclerotic
plaque and heart valves.
The 2.79 µm Erbium:yttrium-scandium-gallium garnet laser (Er:YSGG) was the most effective in
removing calcium without significantly shattering the adjacent tissues161 at settings of 0.2 J per pulse (6
Hz) and fluence of 238 Joules per cm2. Freshly removed aortic valve tissue can be meticulously and
expeditiously debrided using a Thulium-Holmium-Chromium:YAG laser.161 Use of lasers with lower
absorption coefficients for inorganic salts and collagen require higher fluences and results in undesired
collateral thermal and shock wave injury, leading to unacceptable scarring similar to that seen with
ultrasonic and mechanical-manual debridement.162
Laser debridement of acquired calcified stenotic heart valves remains highly experimental, with an
uncertain future.
9.7.2 Myotomy, Myomectomy, and Myoplasty

Catheter-guided laser resection or incision has been suggested as a possible alternative to open heart
surgery. In 1984, Cleveland163 used as an argon laser system to remove an abnormally enlarged section
Applications and Regulatory Approvals

(Oct. 2001)
U.S. FDA European
Coronary angioplasty
Yes Yes
Pacemaker electrode removal
Yes Yes
Lower extremity angioplasty
In clinical trials
(PELA; LACI) Yes
Coronary stent restenosis
In clinical trial (LARS) Yes
FIGURE 9.23 Spectranetics Model CVX-300: This xenon chloride (excimer) laser system has a wavelength of 308
nanometers; it delivers 40 pulses per second at a pulse width of 125 to 200 ns. The output fluence is 30 to 60 millijoules
per square millimeter.
of heart muscle that was obstructing blood flow through the aortic valve, a condition known as hyper-
trophic subaortic stenosis. Conventional surgical treatment for patients with this hypertrophic cardiomy-
opathy who are refractory to medical therapy is to place the patient on an open heart bypass, open the
aorta, visualize the left ventricular outflow tract below the aortic valve, and then to make an incision
into the thickened heart muscle (septal myotomy or myomectomy).164–166
Cleveland’s cardiologist colleague Isner chose the argon laser, first because its wavelength (454-514
nm) is in the visible range so it could be used to illuminate the intraventricular operative field, and
second, the wavelength is well matched to the absorption spectrum of myoglobin, the principal constit-
uent of cardiac muscle. As a result, absorbed light energy is converted efficiently into heat that can initiate
an intense, localized thermal reaction and vaporize the target myocardial tissue into the equivalent of a
myomectomy. A 200 micron fiber coupled to an argon laser was used to deliver 1.5 watts in less than 4
min to create a 4 cm long, 1 cm deep, 0.5 cm wide incision. The patient had an uncomplicated post-op
course and was well 5 years later. This group found no experimental evidence of toxic byproducts or
particulate debris.167
Lasers have also been used to make surgical incisions into the heart muscle (cardiomyotomies) for
other purposes — and for the dissection of the scar tissue that surrounds the heart and great vessels
(aorta and pulmonary artery) during reoperations, in an attempt to limit bleeding and minimize trauma
to surrounding tissues. The clinical experience for these purposes is very limited and consists mostly of
anecdotal reports. Figure 9.23 refers to the laser currently approved for angioplasty.
9.7.3 Septostomy
In certain congenital anomalies, such as transposition of the great vessels, a fatal condition in which
the infant is born with aorta and pulmonary artery recessed, total anomalous pulmonary venous
return, and pulmonary and tricuspid atresia (a form of very severe stenosis or absorption) with intact
septums, surgical correction is often not possible or carries a great mortality in neonates. A palliative
mixing procedure is often done, either a surgical septostomy or a Rashkind balloon septostomy.168
Such procedures may allow the infant to survive for several years and to grow to a size that is compatible
with survival after surgical correction.
Riemenschneider,169 Bommer et al.170,171 and Ben-Sacher et al.172 performed atrial septostomy in human
infant autopsy hearts and in living dogs using an argon laser delivered transvenously by a fiber. They
thought that the method was feasible and could provide a means of performing a septostomy in patients
in whom balloon septostomy is either difficult or not possible, such as older infants or those lacking a
foramen ovale. To date, there are no reports of this theoretically promising technique in patients.173
9.7.4 Endarterectomy
Attempts to restore blood flow through obstructed arteries and veins by removing the obstructing material
had invariably failed prior to dos Santos’ report in 1947174 of a radical procedure in which he removed
not only the occluding arterial atherosclerotic plaque, but all tissue to the internal elastic lamella.
While this procedure, termed thrombo-endarterectomy, is generally considered to mark the advent of
modern vascular surgery, it has largely been replaced by bypass operations with either a vein or a
prosthesis. Nonetheless, thrombo-endarterectomy remains the procedure of choice for certain specific
obstructions, most notably in the carotid artery in the neck.
The healing of endarterectomy wounds created with different lasers was compared in experimental
animals.175–177 Thermal injury with resultant thrombogenesis was readily apparent. It was minimized by
delivering the laser energy in short pulses. In October 1984, Fasano et al. performed the first endarter-
ectomy in a patient. They used an argon laser coupled to a 1 mm fiber to deliver 21 pulses of 3.5 watts
at 0.7 second duration to aid in the removal of a carotid artery plaque in a 51-year-old. There was good
clinical recovery.178
Eugene and associates have reported a careful comparison study of CO2, Nd:YAG and argon and
concluded that argon provides the most satisfactory tissue response.179,180 This group had earlier
attempted to do endarterectomy and weld the endpoints using a xenon chloride excimer laser (308 nm).
They found that, with a 50 mJ/pulse at 120 ns pulse duration and either 15 or 20 Hz frequency, they
could dissect the atheroma quickly, precisely and easily, but they could not weld the end points. These
findings are consistent with the non-thermal mode of action of this excimer laser. The non-thermal
ablation of minute amounts of tissue makes the excimer laser an ideal scalpel, precisely because of its
non-thermal mode of action.181
Eugene’s group has reported on two clinical trials with argon laser light delivered through a 300 µm
quartz fiber at an average power of 1.0 watt. The surgical technique is shown in Figure 9.24. After incising
the artery across the plaque, the fiber is positioned at a distance to make a spot size of approximately
0.5 mm in diameter. For perforation, the beam is positioned perpendicular to the surface; for dissection
of the plaque, the beam is delivered tangentially, and for welding, it is kept moving over the surfaces
while a saline drip is used to cool the tissues.
Eighteen endarterectomies ranging in length from 6cm to 60cm were done in 16 patients’ arteries:
aortic, iliac, superficial femoral, profunda femoral and popliteal-posterior tibial. The atheromatous
plaque was dissected free within the classic plane at the internal elastic lamella. The endpoints were then
FIGURE 9.24 Laser endarterectomy.

welded to the remaining vessel wall by the laser and the incision was closed by welding. Three patients
had early thrombosis requiring thrombectomy. All patients had good clinical results. The ankle–arm ratio
(the ratio of the blood pressure in the ankle to the arm) went from a pre-op mean of 0.53 to 0.97 post
op (normal is considered over 0.8). The 1-year patency was 88%.182
Ten carotid endarterectomies were done in nine patients; all were well clinically and the vessels were
patent on duplex ultrasonic imaging at 1 year.183 Although these results are satisfactory and the
procedure appears to be safe, no real advantage has been shown over the classical surgical endarter-
ectomy. There is great potential value, however, in tacking the endpoints with the laser instead of
mattress sutures, and a tapering of large protruding ledges could reduce turbulence and promote
patency.
9.7.5 Vascular Anastomoses: Laser Welding in the Cardiovascular System

Laser–tissue welding (Figures 9.25 and 9.26) has been investigated for possible advantages of speed, ease
and reduction in inflammatory response. An additional advantage of laser welds is the ability to allow
growth of the anastomosis with the growth of the patient. This is of great importance for vascular repairs
in infants and children. Continuous suture repair does not allow for growth, and even interrupted sutures
may limit growth. Obstacles to acceptance of this new technology are the excellent results of long-
established suture techniques and hemostatic compounds in most cardiovascular applications, and the
added time required to set up to do the laser weld.
The exact mechanism of laser–tissue welding remains uncertain. Theories have ranged from a denial
of even protein denaturation to steadfast support for the formation of covalent bonds as the only means
by which sufficient weld strength could be attained. Most investigators assume that tissue welding is
accomplished by a photothermal mechanism. However, some have speculated that laser light may induce
other effects in tissue independent of heating that are responsible for welding, such as photochemical
bond cleavage or bond formation. Such mechanisms occur in the lens of the human eye secondary to
UV light absorption from sunlight.184
FIGURE 9.25 Technique for performing laser welding of the distal anastomosis of a vein-to-artery bypass. Sutures
are placed at the apices of the incisions and at the middle of the posterior wall (A). Tension on the suture (solid
arrow) at the middle of the posterior wall apposes the edges of the repair for welding (B). A suture is placed in the
middle of the anterior surface of the anastomosis (C) and apposes the edges for welding (D). Broken arrow represents
site of laser energy delivery. (Reprinted from White RA et al., Mechanism of tissue fusion in argon laser welded vein-
artery anastomoses, Lasers Surg Med. 8:83-89, 1988, without permission.
FIGURE 9.26 Laser welding. Schematic of the concept of argon laser–tissue fusion. (a) Apposition of collagen fiber
in a tissue incision. (b) Mild heating of the collagen fibers by laser energy causes unwinding of the helical configu-
ration. (c) Formation of covalent bonds in the annealing tissues with cooling of the site by saline irrigation. (Reprinted
from White, RA and Kopchok, GE, Laser vascular tissue fusion: Development, current status and future perspective,
J Clin Laser Med. Surg 8:47-54, 1990, without permission.
Several investigators have studied the effects of laser welding on structural proteins such as collagen.
Schober et al.185 demonstrated loss of periodicity, increased caliber and interdigitation of collagen
fibrils in rat carotid arteries anastomosed with the Nd:YAG laser. Using a diode laser, Bass et al.186
welded rat tail tendon, type I collagen, the principal structural protein in skin, blood vessels and
connective tissue and observed no novel covalent bonds or cleavage of bonds. However, protein was
denatured. Disulfide bonds are not possible in type I collagen so the role of this type of bonding was
not evaluated. Noncovalent bonding or interaction among denatured collagen molecules is suggested
as the likely mechanism.
Lemole et al. correlated tensile strength of bovine tendon welds with tissue temperature187 by using
water bath heating of the tendons and avoiding the use of a laser. The optimum temperature for welding
in this model was 62°C, suggesting that collagen is denatured, as no covalent bonds are broken. Tem-
peratures high enough to cause protein denaturation in the target tissue are required for tissue welding.
The formation of covalent bonding or disulfide bonding may play a role in weld strength, but adequate
tissue welding can be obtained without the presence of either of these effects. Further definition of tissue
level parameters for tissue bonding is required.
Kung et al.188 studied rat and rabbit vessels with wall thickness that approximately matched absorption
depth of 123 um for the l.9 um laser they chose to use. They delivered 150 mW through a silica fiber
held approximately l mm from the target, which resulted in a spot of approximately 0.7 mm.The linear
delivery rate was about 0.3mm/sec. They found that the acute burst pressures of the welds was the
reciprocal of the vessel radius, suggesting that the product of the weld strength and the optical absorption
depth is constant over the range of the vessel sizes studied. The weld strength for a weld thickness equal
to the optical absorption depth was determined to be 4 x 106 dynes/cm2, which is comparable to the
strength of a sutured anastomosis. This group concluded that an optimal weld can be achieved when the
tissue absorption of the laser is matched to the vessel wall thickness. The l.9 um M~ Raman shifted Nd-
YAG laser therefore seems well suited for small-vessel welding. A laser with greater penetration will cause
spread of heat and a laser with more shallow penetration will not achieve a weld. This study raises the
question of the optimal wavelength for anastomoses of vessels of different thicknesses, such as for an
end-to-side anastomosis.
Broader clinical acceptance and application of tissue welding techniques depend on simplification of
the process and more convincing evidence for its value over conventional suture techniques. If the
procedure can be made easier and less dependent on operator skill and judgment, it might find special
application in laparoscopic surgery. Several trends and advances may contribute to the integration of
laser welding into surgical practice. These include special devices to facilitate and automate welding,189,190
the use of computer controlled laser dosimetry,191 and compact, relatively inexpensive diode lasers. The
development of tissue solders is another advance that makes the welding process more forgiving. The
chosen solder can compensate for defects in tissue apposition and imperfections in dosimetry by absorb-
ing most of the laser energy and protecting the underlying tissues to be bonded. A soldered anastomosis
also reduces the chance of excessive heating and irreversibly damaging host tissues. If initial attempts
with solder fail, reapplication of solder allows repeated trials without thermally damaging the host tissue.
In addition laser–tissue fusions created with fibrinogen and other protein solders are stronger than bonds
formed without the solder.192,193
Use of a dye that absorbs laser energy enhances selective localization of heating with less collateral
injury and allows use of less energy for the weld.194,195 Laser-dye pairs can be chosen so that the laser’s
output wavelength matches the absorption peak of the dye closely, thereby providing efficient and target-
specific laser energy delivery. This use of an exogenous dye is an alternative to trying to find a “best”
universal wavelength for all native tissues, because the dye dominates the laser–tissue interaction regard-
less of water content or endogenous pigmentation.
A tissue solder consisting of human fibrinogen mixed with indocyanine green dye and an 808 nm
solid state diode laser has been evaluated very recently.196–198 No exogenous clotting activators such as
thrombin or calcium are used with the fibrinogen solder. Indocyanine green has a large absorption
peak close to the 808 nm. Another feature of the 808 nm wavelength is that it is within an “optical
window”199 for vascular and other tissue. This wavelength fails to show any tissue effects even at the
highest power outputs available (9.6 W/cm2) unless the indocyanine dye is present. Thus, despite
elevation of the temperature of the dye-enhanced fibrinogen during laser exposure, the underlying
vessel wall is minimally damaged.
These concepts are illustrated in a rabbit aortotomy model.200 The immediate bursting pressures of
welds created without fibrinogen (262 +/– 29 mmHg) were significantly less than repairs with fibrinogen
(330+/-75mm Hg, p<0.05). When exposed to urokinase (25,000 IU), the bursting pressures of repairs
performed with fibrinogen were not significantly different from baseline(290+/-74 mm Hg). Suture-
closed aortotomies did not burst, but leaked at pressures significantly below those of laser-closed vessels
(l65+/-9 mm Hg, p<0.0l). Fibrinogen-soldered arteriotomy repairs in rabbits were examined from 1 to
120 days postop. No anastomotic ruptures, thromboses or aneurysms were identified. Soldered sites
rapidly regenerated a new intimal surface and healed by myofibroblast proliferation. No significant foreign
body response was identified; the fibrinogen was eventually completely reabsorbed. The inability of
urokinase to alter the strength of fibrinogen-soldered aortotomies suggests that the activation of fibrin-
ogen by the normal clotting mechanism is not the dominant mechanism for producing bond strength.201
Several problems complicate the procurement of fibrinogen. Although possible, it is logistically com-
plex to take the patient’s own blood during the preoperative period to produce cryoprecipitate. The use
of homologous plasma products is complicated by the risks of blood infection. In addition, naturally
occurring glues have varying physical properties such as viscosity and stickiness, which complicates the
application. Ideally, a totally synthetic source of the appropriate protein for soldering should be developed.
These difficulties might all be solved by the development of multicomponent glues made of a protein
component, a ground substance component and an energy absorbing dye. By altering the proportions
and characteristics of the components, the physical and optical properties of the glue can be tailored to
specific applications.
9.7.7 Clinical Examples of Tissue Welding

Jain202,203 and Yamagata et al.204 both used Nd:YAG in 1979 to create anastomoses in 0.3 mm- to 1.0 mm-
diameter vessels of animals. Jain claimed that heat caused fusion of collagen within the media and noted
advantages of speed and consistency. After achieving 92.5% angiographic patency for up to 6 months
postop he used this laser to weld a superficial temporal artery to a middle cerebral artery in a patient205
with cerebral artery insufficiency.
Clinical experimentation with CO2, laser welding for the creation of arteriovenous fistula was
initiated in 1985 by Okada et al.206 His first patient was a 44-year-old woman in chronic renal failure,
for whom an arteriovenous (radial artery-cephalic vein) fistula was created using low power
(20–40mW) CO2, laser and a few (typically four) stay sutures. In 1989, Okada et al.207 stated that 35
patients had undergone CO2, laser arteriovenous or arterio-arterial anastomoses without complications
due to the laser, including four who underwent femoro-popliteal or proximal popliteal-distal popliteal
bypass using a saphenous vein graft.
In 1988, John V. White208 used a low-power CO2 laser to create an arteriovenous fistula. Further clinical
experience was obtained by Rodney A. White et al.,209 who reported in 1990 on a series of 10 patients
who underwent forearm Brescia-Cimino arteriovenous fistula creation using the argon laser. These fistulas
were welded using a technique in which four-quadrant sutures divide the anastomoses into four equal
segments of approximately 5 mm in length.210 Each segment required about 75 to 100 seconds to fuse
using approximately 0.5 watts of argon laser power. White’s group used continuous saline irrigation to
prevent tissue overheating. Patients have been followed by physical exam and duplex scanning. After 4.5
years of follow-up, seven of the patients continue to have functioning fistulae without evidence of
hematomas, stenosis or false aneurysms. Three patients required delayed fistula revision, which was
thought to be due to inadequate development of the fistula or proximal vein thrombosis not related to
the laser welding process. Histology of the fistulae at time of revision demonstrated good healing.
A second clinical series is under way combining argon laser fusion and absorbable sutures. White’s
group has also begun a clinical study of laser fusions of the distal anastomoses of femoral-popliteal
bypasses. Tissue glues or solders have recently been applied clinically in a small clinical series of arteri-
ovenous fistulae.211 Blood loss, operative time, and patency were similar to sutured anastomoses. However,
no significant advantages of the lasered anastomoses over sutured ones were identified.
The feasibility of using laser activated tissue solders to reinforce human arteriovenous fistulae created
with PTFE graft material has also recently been demonstrated.212 Such solder reinforcement could be
advantageous in situations in which an anticoagulated patient is bleeding from a site for which
placement of an additional suture is difficult or could be complicated by entrapment of the back wall
of the anastomosis.
These long-term clinical studies have reinforced the experimental observations that laser assisted
vascular anastomoses are capable of excellent long-term healing. The initial strength of the weld, however,
remains a concern, because all of these examples had reinforcement of the anastomosis with sutures. It
is not known if these anastomoses could have been accomplished without the additional immediate
strength provided by the sutures. Bass et al.213 have pointed out that completely sutureless anastomosis
of microvessels is entirely possible with immediate bursting pressures above 300 mm of Hg. This very
high time-zero bursting pressure is probably due, in part, to the favorable consequences of LaPlace’s law,
when applied to vessels of small diameter. From the clinical standpoint, it is in these microvessels that
laser vascular welding makes the most sense, as it is for these vessels that the technical difficulty of placing
sutures is the greatest, and also for which the hemodynamic flow disruption and healing disturbances
caused by sutures have the greatest relative impact.
Although the results of these studies have been generally good, significant obstacles to clinical accep-
tance remain, including the need for precise tissue apposition, concerns over the initial strength of fusion,
difficulty of clinical recognition of the optimum endpoint for energy application, logistical problems
with laser utilization, and limited availability of biocompatible materials to constitute appropriate tissue
glues or solders. Progress in automated welding techniques, refinement and development of better glues
or solders and availability of logistically simple diode lasers should eventually overcome these obstacles.
The FDA recently approved a fiberoptic catheter device manufactured by Cardiofocus for treatment
of chronic atrial fibrillation. The catheter device creates a linear atrial ablation lesion that is an
improvement over radiofrequency ablation catheters in blocking conduction that would otherwise
allow tachyarrythmias to recur.214
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147. Uzun O, et al., Laser valvotomy with balloon valvuloplasty for pulmonary atresia with intact
ventricular septum, JACC 27 (#2, Suppl. A, Feb.) 1996, p.120A.
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150:1647, 1952.
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163. Isner, JM et al., Laser myoplasty for hypertrophic cardiomyopathy. Initial in vitro experience in
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outflow obstruction, J Thoracic Cardio Surg 76:423, 1978.
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Chest 80:550- , 1981.
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168. Rashkind WJ and Miller WW, Creation of an atrial defect without thoracotomy JAMA 196:173,
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10
Lasers in Photodynamic
Therapy
10.1 Introduction ....................................................................... 287

PDT Registration and Drug Plus Device Co-Dependence
10.2 Tissue Optical Properties, Photobleaching,
and Light Delivery Systems................................................ 289
10.3 PHOTOFRIN PDT for Superficial Bladder Cancer......... 291
10.4 PHOTOFRIN PDT in the Treatment of Endobronchial
Lung Cancer........................................................................ 296
10.5 PHOTOFRIN PDT in Gynecologic Malignancies ........... 300
10.6. PHOTOFRIN PDT in Head and Neck Cancer................ 302
10.7 PHOTOFRIN PDT in Gastrointestinal Cancer ............... 304
PHOTOFRIN PDT for Esophageal Cancer • PHOTOFRIN PDT
for Colorectal Cancer • PHOTOFRIN PDT for Early Gastric
Cancer
10.8 PHOTOFRIN PDT for Intraoperative Abdominal
or Thoracic PDT ................................................................ 306
10.9 PHOTOFRIN for Intraoperative PDT in the Treatment
of Intracranial Tumors....................................................... 307
10.10 PHOTOFRIN PDT in the Treatment of Ocular Cancer . 310
10.11 PHOTOFRIN PDT in the Treatment of Cutaneous
and Subcutaneous Tumors ................................................ 311
10.12 New Photosensitizers for PDT .......................................... 313
VISUDYNE™ (Verteporfin) • LEVULAN® (Aminolevulinic
Stuart L. Marcus, M.D., Acid HCl, ALA) PDT
Ph.D. 10.13 Conclusions ........................................................................ 317
DUSA Pharmaceuticals, Inc. References ...................................................................................... 317
10.1 Introduction
Photodynamic therapy (PDT), a subset of photochemotherapy, is a therapeutic method requiring a drug
(photosensitizer), a device (light source and light delivery device), and the presence of molecular oxygen
for the treatment of a variety of human clinical conditions.1–3 Clinical photodynamic therapy (PDT) is
a two-stage process, with the first stage consisting of the delivery of a photosensitizer, either systemically
or topically. The first photosensitizers used historically for this purpose were hematoporphyrin derivative
(HpD, PhotoFRIN I) and porfimer sodium (PHOTOFRIN, formerly known as dihematoporphyrin
ether/esters, DHE, or PhotoFRIN II). PHOTOFRIN (QLT, Inc), is a partially purified preparation of the
photoactive ingredients within HpD, with a proportionate increase in potency. The only reported repro-
ducible side effect of drug injection is cutaneous (skin) photosensitivity, which lasts approximately 4–6
weeks. Following a period of time during which PHOTOFRIN is mostly cleared from a variety of tissues
0-8493-1146-2/02/$0.00+$1.50
(48–72 h) and retained in tumor, skin, and organs of the reticuloendothelial system (including liver and
spleen), the tumor is illuminated with 630 nm wavelength light, a process that constitutes the second
and final stage in the therapy.1–3
PDT-induced target tissue destruction requires the presence of oxygen and is thought to be due to the
production of singlet oxygen for direct cytotoxic effect; a microvascular shutdown effect is also observed
that results in tumor death from oxygen starvation (ischemic necrosis).1,4 Vascular endothelium may
selectively take up HpD or PHOTOFRIN within tumor tissue.5,6 Laser Doppler measurements were
performed to determine blood flow velocity in normal and in tumor blood vessels in mice bearing
spontaneous mammary tumors and in rats with chondrosarcoma (cartilage tumor) implants.7 PHOTOF-
RIN was administered to the tumor-bearing animals, followed 24 h later by light exposure. Decreases in
blood flow occurred within 10 sec of light treatment and were maximal within 5 min.7 The rapidity of
response suggests a thrombotic phenomenon, and in a recent report,8 thromboxane was detected sys-
temically in rats given PDT within minutes of light exposure. Tumor selectivity in treatment is thought
to occur through a combination of selective retention of PHOTOFRIN selective delivery of light, and
light dose selection such that normal (non-tumor) tissue is clinically unaffected.1–3 PDT toxicity is local,
and dependent upon the organ being irradiated.
Fluorescence produced by photosensitizers has been used as a means of detecting tumors or defining
tumor margins.9,10 This use of photosensitizers will not be covered in this chapter, which deals solely
with therapy. This chapter will primarily discuss those patient treatments using PHOTOFRIN and HpD
as prototype photosensitizers for PDT. Devices in development which may render the application of PDT
easier or more user-friendly will also be discussed. Following this discussion of PDT using PHOTOFRIN
or HPD, newer approved photosensitizers such as VISUDYNE™ (verteporfin for injection), and LEVU-
LAN® (aminolevulinic acid HCl) will be briefly discussed.
10.1.1 PDT Registration and Drug Plus Device Co-Dependence

Clinical drug development for anticancer drugs usually proceeds in three phases. The first clinical
experiments in man, termed Phase I studies, are primarily concerned with drug safety. In Phase I studies,
the maximum tolerated dose of drug- and dose-limiting side effects are determined in a small number
(approximately 20–50) of human subjects. Phase II studies target specific diseases for potential therapeutic
effect by the new drug, and serve to separate drugs of genuine potential from drugs that are inactive in
specific cancers or that are very toxic in specific patient populations. Phase II studies may involve between
100 and 200 cancer patients, although they may be larger if many tumor types are to be screened as
potential targets. Once efficacy in therapy is shown, the drug may be compared with standard therapy
(if a comparator drug has been approved and is available) in large clinical trials that might involve
hundreds of patients (Phase III studies). In such studies, the patient is usually randomized to the
“standard” vs. “experimental” therapy. The Phase III study is the most rigorous and extensive type of
scientific clinical investigation of a new drug or therapy.
PDT with HpD or PHOTOFRIN has been reported effective in publications citing thousands of
patients treated.3
The unique characteristics of PDT have made the registration process complex for various reasons.
In countries such as the U.S., a combined drug and device (in this case specific fiber optic entities)
product license filing application must be prepared, and may require review by a combination of agencies
that normally deal with reviewing either a drug or a device. It is important to note that, despite the large
number of anecdotal reports on the clinical success of PDT, no matter how large a patient series by an
original clinical investigator is published, such publications are often viewed as “multiple clinical anec-
dotes.” Such anecdotal reports are usually not acceptable as data for registration purposes unless objective
evidence of patient condition, treatment methods, and response as well as information on the safety of
the investigational agent is provided.
Three photosensitizers have now received marketing approval for the treatment of diseases in the
United States. In 1995, PHOTOFRIN was the first PDT agent approved for the palliation of malignant
Lasers in Photodynamic Therapy 289
dysphagia caused by esophageal cancer. Since then, it has also been approved for the treatment of recurrent
superficial endobronchial lung carcinoma. In 1999, an endogenous photosensitizer, LEVULAN® (ami-
nolevulinic acid HCl, DUSA Pharmaceuticals Inc, marketed by Schering AG) became the first PDT agent
approved for topical use, together with blue light, for the treatment of actinic keratoses (precancerous
skin lesions) in the United States. Most recently, in 2000, VISUDYNE® (verteporfin, QLT Inc) was
approved in the United States as the first drug therapy for age-related macular degeneration, a common
cause of blindness.
10.2 Tissue Optical Properties, Photobleaching, and Light

Delivery Systems
Upon the absorption of light by a photosensitizer, energy is passed (in a nonradiative process) to an
oxygen molecule, increasing the energy state of the oxygen molecule from a triplet configuration to the
excited singlet state. The singlet oxygen may react with many cellular molecules, and interaction with
the plasma and mitochondrial membranes is currently thought to be the method of cytotoxic action of
the photodynamic effect.2
The optical properties of tissue, for light propagation at the 600 to 1100 nm wavelength range, are
dominated by scattering effects (see Ref. 2 for review). Thus, the propagation of light in tissue is best
described by diffusion theory using the scattering coefficient, ä, and the total attenuation coefficient á,
which includes tissue absorption as well as scattering. It is to be noted that both ä and á are wavelength
dependent, and that absorption varies significantly among various tissues that contain differing chro-
mophores (mainly hemoglobin and melanin).2
One of the findings that will be notable throughout the clinical section of this chapter is the selective
toxicity of PDT for tumor tissue. While this can be explained, in part, by selective retention of PHOTOF-
RIN within the tumor, a phenomenon termed photobleaching appears to also provide an effective ampli-
fication of tumor selectivity. Photobleaching is the permanent photochemical loss of the chromophore
used in PDT. If the photosensitizer used in PDT were completely stable and could therefore pass energy
to oxygen molecules as long as light was present, the maximum depth of tumor necrosis that could be
achieved without damaging normal tissues would be determined entirely by the ratio of drug in tumor
to that in surrounding (and also irradiated) normal tissues. If the concentration of drug in normal skin
surrounding a tumor is one third the concentration in tumor, and the photobleaching rate is the same
for both tissues, then the maximum photodynamic dose in normal skin is one third that of the tumor
and no amount of light will damage the normal skin but the tumor will undergo necrosis. Dougherty
and Marcus2 have shown examples of the clinical application of this concept in the treatment of skin
tumors.
The wavelength recommended for activation of PHOTOFRIN by light is 630 ± 3 nm using mono-
chromatic, coherent light (laser light), although light of shorter wavelengths can also activate the drug.
The 630 ± 3 nm wavelength was chosen because it is the longest wavelength capable of activating
PHOTOFRIN and, therefore, has the deepest tissue penetration (3 mm to 8 mm)3,4 for use of this
photosensitizer. Non-laser light sources have been used to activate PHOTOFRIN but lasers are preferred
for applications requiring endoscopic delivery of light into the body because the properties of laser light
provide efficient coupling into small optical fibers.
The fiber optic tips used for the delivery of laser light in preregistration studies using PDT with
PHOTOFRIN in North America and Europe are shown in Figure 10.1. The microlens-tipped fiber optic
provides a uniform field of light for front surface illumination. Cylindrical-diffuser-tipped fiber optics
provide approximately 95% of diffused light in a uniform, cylindrical distribution perpendicular to the
axis of the fiber tip; these fibers can be used intraluminally (placed within the lumen of a cylinder-shaped
organ part) for delivery of light to the wall of a hollow viscus such as the bronchus or esophagus when
it is not possible to point a microlens-tipped fiber directly at a lesion. Five lengths of cylindrical tips are
provided, from 0.5 to 2.5 cm, in increasing increments of 0.5 cm. A tumor longer than a cylindrical tip
FIGURE 10.1 Examples of fiber optics used to deliver laser light for photodynamic therapy. (top to bottom) Spherical
light diffuser for isotropic light delivery to spherical or near spherical body cavities; microlens-tipped fiber for uniform
front surface illumination; Cylinder diffusers for delivery of light intraluminally or interstitially; 0.5-cm length pointed
cylinder diffuser; 1.0 cm length pointed cylinder diffuser; 2.5 cm length pointed cylinder diffuser. Reprinted from
Ref. 3 with permission of the publisher.
may be treated in non-overlapping segments using different sized fiber optics in sequence. The cylindrical
diffuser tipped fibers are prepared with pointed ends, so that they can also be inserted directly into the
tumor substance for efficient, tumor-specific interstitial light delivery, particularly for thick lesions. The
spherical diffuser-tipped fiber optic provides an isotropic, spherical distribution of light and is used for
PDT of the entire urinary bladder, but could be applied to any indication where a spherical or near-
spherical distribution of light is required. Modifications of these general fiber optic delivery systems will
be discussed under the specific category of malignancy for which they have been developed.
Lasers necessary for producing light to be delivered via fiber optic devices must be considered in plans
for product license applications. In the Phase III studies carried out and sponsored by QLT, Inc. within
North America for PHOTOFRIN the only laser type used is the argon ion laser-pumped tunable dye
laser (APDL). The continuous-wave output APDL may be contrasted with pulsed output lasers such as
the gold vapor laser and the copper vapor laser-pumped dye laser.11 These lasers have pulsed outputs in
the 10- to 100-kW range, although power is usually presented as average output power and they have
been used in a fashion identical to the APDL. The excimer laser-pumped dye laser system has been used
extensively in Japan, and this was the laser system used for some Phase III registration studies in Japan.
The excimer-pumped dye laser is an extremely high-power pulsed laser system in which peak power may
approach the megawatt range. There have been no published controlled studies comparing clinical PDT
efficacy and safety results in the APDL system with pulsed-output laser systems. By comparing the safety
and efficacy of APDL lasers with copper vapor laser-pumped dye laser systems in otherwise identical
clinical trials for lung and bladder cancer in different European centers, data will be obtained that can
be used to determine whether such laser systems are indeed equivalent.
Solid state laser systems such as the frequency-doubled Nd:YAG laser-pumped dye systems or visible
diode lasers are now being used in PDT systems. Advancements in diode technology have now allowed
the production of relatively inexpensive lasers emitting in the wavelength range of 630–700 nm at powers
currently considered therapeutically useful, in the 1- to 4-watt output range. The availability and appli-
cability of new diode lasers to PDT is discussed in later sections of this chapter.
Light delivery systems as they currently exist for PDT, although adequate, may be modified in the
future to allow in situ dosimetry of delivered light. Because of the geometry of certain organs or the size
of areas to receive light for photosensitizer activation, current methods of fluence determination are
unquestionably suboptimal. Devices that have been developed to provide such in situ methods of dosim-
etry have been developed for PDT of the entire urinary bladder (see next section) and for PDT of
intrathoracic and abdominal spaces. The potential advantage of such systems would be to prevent under-
or overtreatment of target organs with activating light. Discussions of such needs and those applications
of PDT in which in situ dosimetry might be most appropriately be utilized will be discussed in sections
dealing with specific cancer treatment.
In each of the following sections, the characteristics of each cancer (incidence, prognosis), together
with current standard methods of therapy and results of such therapy, will be briefly discussed. The
current status of PDT in the treatment of each particular form of cancer will be critically reviewed as
well as any technical advancements that might increase the potential for PDT effectiveness.
10.3 PHOTOFRIN PDT for Superficial Bladder Cancer

Superficial bladder cancer will be diagnosed in approximately 50,000 patients in the United States during
2001. Such disease can present as papillary tumors (tumors presenting as small nipplelike or frond-like
processes) or as carcinoma in situ (CIS). The standard of therapy for superficial papillary tumor treatment
is surgical excision performed via endoscopy through the urethra (a transurethral resection of the bladder
tumor or TUR-BT). Because the penetration of 630 nm light at treatment power can approach 5 mm in
certain tissues, PDT is theorized to be able to eradicate most, if not all, superficial bladder cancer, which
can extend into the mucosa or submucosal layers, but not into the muscular layers of the urinary bladder.
Nseyo et al.12 have recently reviewed the use of PDT in this disease entity, and the results of clinical trials
published in the literature13–26 are summarized in Table 10.1. The reports each include between five and
34 patients, some of whom were treated for multiple lesions. Whole bladder PDT, in which light is
delivered via a spherical diffuser-tipped fiber optic centered within a fluid-distended bladder, is generally
used for treatment of CIS or for the prevention of tumor recurrence following papillary tumor removal.
Focal therapy using microlens-tipped fiber optics to deliver light to papillary tumors has also been used.
Partial or complete removal of papillary tumors occurred in 70–100% of sites treated (Table 10.1).
Although the incidence of side effects in bladder cancer PDT is not always reported, there appears to be
a general “post-PDT syndrome” characterized by irritative bladder symptoms of urinary frequency,
urgency and dysuria (burning feeling on urination) that is transient but that may vary in duration, from
a few days to several weeks.12 Irreversible bladder contraction has been reported occasionally. Such results
appear to be due to high light dosing, bladder overdistension, or vigorous treatment of patients already
having fibrosis (scarring) as the result of prior radiation therapy.12,25
In those patients who received focal tumor illumination, the severe toxicities reported by some authors
may be due to lack of correcting for backscattered light reaching the entire bladder, which could have
raised the total light dose to the whole bladder from 50% to 300%, depending upon the number of focal
lesions treated. Alternatively, the drug and light doses that might prove optimal in terms of efficacy and
TABLE 10.1 Summary of Published Results with PDT in Bladder Cancer
292
Therapy Patient Response (lesions

Patients/ Description of Drug Dose Patient Follow-up
Reference Lesions Disease (mg/kg) PDT Procedures CR PR NR RE (months)
Benson [13,14] 27/31 15-focal CIS HPD 2.5 150 J/cm Flat tip with 15 0 0 8 6 to 32
12-diffuse microlens 12 0 0 2 (mean 7)
CIS
2-T2 HPD 4 to 5 25 to 45 J/cm2 Bulb diffuser 0 0 2 0
2-Ta 3 or 48 h NS 150 to 250 0 0 2 0
mL
Ha et al. [15] 6 Transitional HPD 5 5 1 0 -* 1
cell
Hisazumi et al. 9/48 Ta-T1 HPD 2 to 3.2 150 to 300 1
[16,17] <1 cm mW/cm2
1–2 cm 100 to 200 J/cm2 200 mL NS I

2-3 cm flat tip
>3 cm
22 6 5 -
Nseyo and 10/13 4 TIs PHOTOFRIN 2 10 to 69 J/cm2 Bulb diffuser; 4 0 0 - 2 to 12
Dougherty [18] water or NS;
1 Ta filling press 1 0 -
6 T1 30-60 cm H2O 2 2 -
2 T2
Shumaker and 16 12TIs PHOTOFRIN 2 < 20 J/cm2 NS150 MI 7 2 0 - 6 to 24
Hetzel [19] 4Ta 2 2 6 to 15
Tsuchiya et al. [20] 8 Ta-T2 HPD 2.5 120 to 260 J/cm2 Flat tip; 8 0 0 2 6 to 18
G HI
Nseyo et al. 23 6 TIs PHOTOFRIN{ 2 16 to 200 J/cm2 Bulb diff. 4 1 1 2 to >12
[21,22] 8 Ta Microlens or 3 3 2 2
8 T1 Cylinder 2 5 1
3 T2 0 1 2
1 T3 0 0 1
Prout et al. [23,24] 20/50 3 TIs PHOTOFRIN 2 5.5 to 10J/cm2 Bulb diff. 3 0 0
Microlens
50 Ta 100- 12 25 13 3
or T1 200J/cm2
Lasers in Medicine
Harty et al. [25] 7 3 Ta PHOTOFRIN 2 100 J/cm2 focal Microlens 3 0 0 1
2 T1 +25 J/cm2 whole Bulb 1 0 1 0
2 TIs bladder Diffuser 1 0 1 0
Dugan et al. [26] 24 21 Ta,3T1 PHOTOFRIN 2 12 patients
after TUR 15 J/cm2 whole
bladder
spherical bulb Median time to recurrence 93 days — observation arm
diffuser
Not reached for PDT arm

12 patients
observed
CIS = carcinoma in situ NR = no response

Lasers in Photodynamic Therapy
CR = complete response NS = normal saline

PR = partial response RE = recurrence
TIs, Ta, R1, T2 = clinical stage of bladder cancer from CIS to superficial muscle invasion
* Data not provided.
293
safety may not have been deduced during clinical trials. None of the studies listed were true Phase I
studies, in that careful stepwise elevation of light dose (starting at a low dose of photoensitizer) was not
attempted. Therefore, the clinically “optimal” dose for drug and light has really never been determined
in this indication.
The severity of toxicities observed in PDT may also vary according to the amount of bladder mucosa
involved with disease, as well as with the position of the light delivery device. Centering of the intravesical
light delivery device within the bladder must be accomplished to avoid undertreatment or overtreatment
of differing mucosal areas. In Phase III protocols (see below), centering of spherical diffuser-tipped fiber
optics was specified by both sounding and ultrasound-directed methods. Two devices have been rede-
veloped, each for the dual purpose of centering the fiber optic and determining in situ light dosimetry.27,28
The device described by Marynissen et al.27 consists of a modified cystoscope and three translucent nylon
catheters that are unfolded inside the bladder and into each of which is inserted a dosimetry probe
(Figure 10.2). As bladder shape is not normally spherical, equalizing the light output reaching each probe
provides a method of centering the light source as far as can be possible. In situ dosimetry also provides
a method of determining real-time changes in the light reaching the bladder wall caused by variation in
laser output or changes in the transparency of the intravesical fluid due to bleeding or mucosal sloughing.
However, this device requires a modified cystoscope and considerable set-up time, as a) essential pre-
forming of the nylon catheters is accomplished outside the bladder after determining the neck-to-dome
distance and b) light must be checked on three devices as the detectors are moved through the catheters
across the entire bladder wall.
Nseyo, et al.28 have obviated the need for multiple detectors for fiber optic tip centering by using an
intravesical laser catheter (IVLC) delivery system to convert a bladder into as near a spherical organ as
possible (Figure 10.3). The IVLC consists of a translucent balloon that is inflated with fluid to produce
spherical distension of the bladder; a central lumen within the catheter automatically centers the fiber
optic tip. A light sensor on the IVLC monitors light reaching the bladder wall and displays real-time
irradiance (mW/cm2) as well as accumulated light energy (J/cm2) and can be used to automatically
monitor and adjust treatment time based on changes in light fluence. Outlet ports in the device allow
removal of urine produced during the procedure, thus keeping the bladder distended to a constant
volume, and provide a means of irrigation to remove blood or other light-interfering secretions without
altering bladder volume. Although devices such as the IVLC appear to increase ease of whole-bladder
PDT, the effect of such devices on the safety of whole bladder PDT awaits results of their testing in
clinical trials.
A phase III controlled clinical trial of whole bladder PDT for the prophylaxis of recurrent superficial
bladder cancers (papillary type) has been carried out.26 After transurethral resection (TUR) of bladder
tumors (up to stage T1G3), patients were randomized to either a single course of PDT using PHOTOF-
RIN and a whole bladder light dose of 15 J/cm2 or observation. Follow-up cytologies and cystoscopies
were rigorously carried out every 3 months. An unscheduled interim analysis performed after accrual
of 24 patients well matched in demography after a median follow-up of 116 days revealed that the
median time to recurrence for the observation group was 93 days but had not been reached for patients
receiving PDT (p <0.001).26 These results are encouraging, for no agent has been approved for prophy-
laxis of bladder cancer and no agent appears to provide efficacy similar to PDT with PHOTOFRIN
following a single intravesical treatment. These data26 formed part of a product license package that
resulted in the approval of PHOTOFRIN for the prophylaxis of superficial bladder cancer by Canada’s
board of health in 1993.
Long-term prophylaxis of tumor recurrence in PDT may be the result of macrophage and lymphocyte
stimulation within the bladder. Preliminary studies have shown that cytokines such as interleukin-1 and
tumor necrosis factor appear in the bladder following PDT (but not TUR or infection) and may be
detected in urine as long as 2 months after PDT even though the patient is asymptomatic.28
CIS that is refractory to at least two courses of intravesical (applied directly to the bladder) chemo-
therapy or immunotherapy has a strong potential for life-threatening muscle or prostate invasion and
subsequent or concurrent metastasis.3,12 Data from six studies reported from 1983 to 1988 have been
FIGURE 10.2 Device developed by Dr. W. Star for centering of fiber optic within the urinary bladder and for in situ
dosimetry, 27 (a) dosimetry unit. (b) Cystoscope with telescope inserted via dosimetry unit for inspection of unfolded
catheters. (c) Telescope replaced by light source for positioning and irradiation (inset = magnified view of light
detector in place within catheter). Figure originally published in. Ref. 27 and reprinted with permission of The Journal
of Urology.
FIGURE 10.3 Line drawing of intravesical laser catheter described by Nseyo et al.28 8 = catheter outlet port. Reprinted
from. Ref. 28 with permission from The Journal of Urology.
reviewed (Table 10.1).3 In the initial studies of 47 patients with refractory CIS reported in the literature,
46–47 (97.8%) were reported to have a complete response (negative cytology or biopsy) with follow-ups
from a median of 3 months to 12 months. More recently, the results of PDT with PHOTOFRIN for
refractory CIS have been reviewed from articles in which 215 patients received from 1.5 to 2 mg/kg
PHOTOFRIN via the intravenous route, with an overall response rate of 66%. Estimated time to recur-
rence in the few large studies with long-term follow-up ranged from 37 to 84 months.29
10.4 PHOTOFRIN PDT in the Treatment of Endobronchial

Lung Cancer
Non-small-cell lung cancer, which occurs in approximately 120,000 patients in the United States annually,
often produces symptoms arising from that portion of the tumor present within the airway (endobron-
chial or endotracheal component). Symptoms may present as hemoptysis (coughing up of blood), cough,
or dyspnea (shortness of breath), the latter caused by direct obstruction of the airway by the tumor
within the airway (endobronchial obstruction) or by compression of the airway by tumor outside of the
airway (extrinsic obstruction).
Symptoms caused by endobronchial tumor obstruction are currently treated with the Nd:YAG laser.
Because the Nd:YAG laser produces a nonspecific thermal laser beam to vaporize or coagulate tumor
tissue, removal of tumor to the bronchial wall would risk perforation. Therefore, only part of the tumor
can be removed using this therapy, although it can be sufficient to improve ventilation. Patients with
recurrent endobronchial lung cancers who, because of concurrent physical conditions such as emphysema
or insufficient lung reserve, cannot be treated at such an early stage with the ND:YAG laser. However,
because of the selective nature of PDT, such patients theoretically can achieve complete endobronchial
tumor removal (complete response or CR) using PDT.
Since the early 1980s, more than 500 patients have been reported in the literature with endobronchial
lung cancer treated with PDT using either HPD or PHOTOFRIN The results of various investigators30–43
are remarkably consistent in that complete and partial response rates ranged from 70% to 100%
(Table 10.2). Best results were obtained with early stage lung cancers. 33,36,38,44 HPD was used at a dose
of 2.0 to 5.0 mg/kg intravenously and PHOTOFRIN at a dose of 2.0 mg/kg intravenously. Laser light
doses ranged from 20 to 500 J/cm2, with power intensity at the fiber tips ranging from 20–800 mW.
One is struck by the wide range of light doses with which at least a partial response can be achieved.
However, as no standardized method of power measurement was practiced among the various insti-
tutions, the consistency of response in the presence of widely varying light doses must be interpreted
with caution.
It is important to note that, in certain studies, patients in very poor condition because of respiratory
distress due to endobronchial obstruction were treated with PDT.45,46 None of the patients died as a result
of therapy and 80% improved clinically, suggesting that the modality might be safe to use in patients too
sick to be considered for the usual clinical trial population. In the studies in which disease cure was the
goal of therapy, follow-up of response ranged from 3 days to 41 months, with the average duration of
follow-up approximately 12 months. The first patients with early disease who were inoperable have now
been reported surviving more than 5 years with no other treatment but PDT.47,48 When symptomatic
improvement or improved operability was the therapeutic endpoint, a positive response was noted in
106 of 111 patients following PDT treatment44-52 (Table 10.3).
All responses are ideally characterized on the basis of a local (i.e., endobronchial site of treatment)
response, with a complete response (CR) defined as the absence of bronchoscopically visible tumor.
Partial response (PR) is defined as a decrease of ≥ 50% in intrinsic obstruction for the tumor site treated.
Stable disease (SD) is defined as decreases or increases in intrinsic obstruction insufficient to qualify as
either a PR or progressive disease. Progressive disease (PD) is defined as an increase in intrinsic obstruc-
tion ≥ 30% attributed to endobronchial tumor growth as compared with the percent intrinsic obstruction
at the time of best response.
TABLE 10.2 Summary of Published Clinical Studies Using PDT in Lung Cancer
Condiction (no. Follow-up Response

Reference Patients/Lesions patients) Drug (mg/kg) Method (mos) CR PR NR Died Side Effects
Cortese and 19/20 Resected or HPD S/1 8-34 6 7 6 9 Phototoxicity, cough

Kinsey [31,32] SC 17 inoperable 2.5 to 5.0 obstruction
Edell and Cortese 38/40 Resected or HPD S 6 days to 14 15 11 25 1_ burn

[33] SC 37 inoperable 2.0 to 5.0 56 mos. Hemoptysis
SCC 1 Obstruction
Cylindroma 1
Fibrosarcoma 1
Hayata et al. [31] 14/20 Early stage (1) HPD S * 1 0 0 — Phototoxicity

SC 6 Stage I (1) 2.5 to 4.0 0 1 0 — 1_ burn
AC 2 Stage II (0) 0 0 0 —
LC 3 Stage III (8) 1 8 0 —
SCC 2 Stage IV (3) 2 6 0 —
metaplasia metaplasia (1) 1 0 0 —
Hayata et al. 21 Early stage HPD S/I Phototoxicity
[31,36] SC 20 unresected (8) 13-41 8 0 0 0 1_ burn
LC 1 resected (5) 7-30 2 3 0 2 possible PDT
Stage I (8) 4-41 2 6 0 4 complications
Kato et al. [37] 43/54 23 obstructed HPD S/I 36-phototoxicity
SC 29 early stage (3) 2.0 to 5.0 3 0 0 — 2 obstr. pneumonia.
Kato [38] AC 5 Stage I (4) 1 3 0 — 2 bronchial fistula
LC 4 Stage II (8) 0 8 0 — 11/23 cleared
SCC 2 Stage III (16) 1 15 0 — obstruction 6/9 pts
metaplasia 2 Stage IV (10) 0 10 0 — operable after PDT
other 1 met (2) 2 0 0 —
Keller et al. [39] 15 Obstructed HPD S/I 17 3 11 1 —
2.0 to 3.0
PHOTOFRIN{
1.5 to 2.0
continued
297
TABLE 10.2 (CONTINUED) Summary of Published Clinical Studies Using PDT in Lung Cancer
298
Condiction (no. Follow-up Response

Reference Patients/Lesions patients) Drug (mg/kg) Method (mos) CR PR NR Died Side Effects
Li et al.[40] 21/24 All advanced HPD 5.0 S 3-7 3 17 4 1 Fever, hemoptysis,

SC 18 progression of in 8
AC 2
SCC 1
Vincent et al. [41] 17 Recurrent, HPD S/I 5-210 0 13 4 8 Fever, pneumonia,
SC 11 advanced 2.5 to 3.0 days hemoptysis
Vincent and AC 5 obstructed (mean candidiasis
Dougherty [42] 40)
LC 1 hypersecretion
Karanov et al. [43] 12 2 Stage II HPD 5.0 1-6 7 2 3 —

10 Stage III
CR = complete response SC = squamous cell SCC = small cell

PR = partial response AC = adenocarcinoma S = superficial
NR = no response LC = large cell I = interstitial
* Data not provided
TABLE 10.3 Summary of Published Studies Using PDT for Palliation or Improved Operability of Lung Cancer
Patients/ Condition Follow-up

Reference Lesions (no. patient) Drug (mg/kg) Method (mos) Results Died Side Effects
Balchum and 81/135 Stable, good HPD S/I to 20 cleared or 80/81 38 cancer 5 pulmonary
Doiron [44] SC 56 prognosis; 72 3.0 improved 6 other hemorrhage
AC 5 obstructed 3 staph pneumonia
LC 4
SCC 4
other 5
metastatic 7
Lasers in Medicine
Hugh-Jones and 15/15 All advanced HPD S/I to 24 CR-3 PR-9 9 cancer 2 burns
Gardner [46] SC 15 symptomatic 3.0-4.9 80% Clinically 2 obstruction
DHE improved
1.9-2.3
Kato et al. [49] 15 Preoperative HPD S/I *11/15 improved 3 cancer
SC 11 therapy 2.5 to 5.0 4 other
AC 2 Stage I (5)
LC 2 Stage II (2)
Stage III (7)
Stage IV (1)
Lam et al. [50] 5/5 All symptomatic DHE S/I 3 to 10 = 100% All CR + PR 1 cancer
* 2.0 clinically improved
followed by
radiation therapy
McCaughan et al. 18/26 Obstructed HPD S/I 0.5–34 CR-12 14 cancer 2 burns
[45] SC 10 symptomatic 3.0 PR-14 61% 2 other 1 obstruction

AC 3 Stage III (15) PHOTOFRIN{ clinically improved 1 pneumonia
LC 1 CIS (1) 2.0 1 hemoptysis
other 4 metastatic (2)
Pass et al. [51] 10/15 Obstructed 6 PHOTOFRIN{ S/I 1-10 CR-2 PR-11 6-cancer 2 obstruction
Lung*4 symptomatic 2.5 MR-2 6/10 1
clinically improved pneumothorax
Metastatic 11
LoCicero et al. [52] 10 Obstructed all PHOTOFRIN{ S/I 1–6 10/10 clinically 10-cancer 2 burns
symptomatic 2.0 improved 1 mild anasarca
CR = complete response I = interstitial AC = adenocarcinoma

PR = partial response CIS = carcinoma in situ LC = large cell
MR = minor response SC = squamous cell SCC = small cell
S = superficial
* Data not provided
299
Palliative randomized, controlled Phase III studies were carried out in North America and Europe to
determine the safety and efficacy of PDT in the treatment of endobronchial lung cancer. Patients (gen-
erally at an advanced stage of lung cancer) presenting with symptoms of dyspnea, hemoptysis or cough
referable to a specific endobronchial lesion were randomized to receive either Nd:YAG laser thermal
ablation therapy, or to PDT with PHOTOFRIN. The majority of patients receiving PDT had light delivered
via interstitial light treatment, which consists of the direct insertion of a fiber optic tip into the tumor
mass and includes point insertion (PI, using a flat-cut fiber tip), point insertion via hollow bronchoscopic
needle for tumors too hard to be penetrated directly by fiber (PIN), and insertion of a cylindrical diffusing
tip (see Figure 10.1), (cylindrical insertion, CI). Intraluminal light treatment consists of the superficial
(or surface-only) photoradiation of tumors, carried out via cylindrical (CS) microlens-tipped fiber
(Figure 10.1), or forward surface (FS) illumination using a flat-cleaved fiber tip. A total of 211 patients
were randomized (102 to PDT, 109 to Nd:YAG) in these studies and, after the first month of follow-up,
objective tumor response was observed in 60% of the PDT-treated patients vs. 41% of the Nd:YAG-
treated patients. Atelectasis improvement in the same follow-up period was observed in 35% of the PDT-
treated patients vs. 20% of the Nd:YAG-treated patients. Clinically significant symptom improvements
were observed that were equivalent in both treatment arms for symptoms of dyspnea and cough; at
month 1 or later, improvement in hemoptysis was seen in 79% of the PDT-treated patients vs. 35% of
the Nd:YAG-treated patients. These results helped support the marketing approval for PHOTOFRIN
PDT for palliation of symptoms caused by endobronchial obstruction in the United States in 1995.53 On
the basis of three noncomparative studies that accrued a total of 62 inoperable patients, PHOTOFRIN
PDT has also been approved for the treatment of microinvasive endobronchial non-small-cell lung cancer
in patients for whom surgery and radiotherapy are not indicated. For these patients, the biopsy-proven
CR rate 3 months after treatment was 50%, median survival was 2.9 years and median time to tumor
recurrence was 2.7 years; disease-specific survival was 4.1 years.53
It appears that PDT has the potential to palliate patients with endobronchial obstruction in a manner
similar to the Nd:YAG laser. However, because of the necessity of waiting 2 to 3 days for selective retention
of PHOTOFRIN patients with acute onset of severe shortness of breath at rest may best be served by
immediate Nd:YAG laser therapy, followed perhaps by PDT to attempt complete removal of the endo-
bronchial obstruction. It is probable that patients with recurrent lung cancer of a superficial nature can
have excellent long-term results using PDT alone, if surgery is not indicated or not possible due to their
physical condition.
10.5 PHOTOFRIN PDT in Gynecologic Malignancies

Gynecologic malignancies include cancers of the vulva, vagina and cervix, all of which are accessible
to endoscopic or surface treatment with light for PDT. Vulvar and vaginal cancer together account
for a total of 5% of female malignancies. Preferred treatment for superficial cancer of both areas
(disease that does not penetrate deeply into tissue and does not involve local lymph nodes) is surgery.
Although invasive cancer of the cervix affects approximately 15,000 women annually, superficial
cervical cancer (carcinoma in situ) is annually diagnosed in approximately 50,000 women and can
be treated by conization (removing a cone-shaped area of the cervix with the cervical opening at the
center of the large surface of the cone). Cryotherapy and CO2 laser surgery have been used in selected
patients with carcinoma in situ of the cervix. Complications of such therapy include scarring, unac-
cepable cosmetic effects, and, in the case of conization, narrowing of the cervical opening affecting
fertility or delivery.
Clinical reports describing the use of PDT with HPD or PHOTOFRIN for gynecologic cancer are
summarized in Table 10.4. Patients had either primary gynecologic cancer or metastatic lesions from
other primary tumor sites. All patients were treated with HPD with a dose ranging from 2.3 to 5.0 mg/kg
or PHOTOFRIN (2.0 mg/kg) and patients were often retreated if the initial response was incomplete.
One researcher administered 20 or more treatments to two patients with local endometrial and ovarian
cancer, and used a Kodak projector lamp as the light source rather than an argon-pumped dye laser. 59
TABLE 10.4 Summary of Published Results with PDT in Gynecologic Malignancies

HPD Dose Follow-up
Reference Patients (sites) (mg/kg) Response (months) Comments
Corti et al. [56] 11 cervix, HPD 2.5 CR PR NR 3 to 25 7/8 patients with

+ 2 uterus, 8 6 1 superficial disease had
+ 2 rectal a CR
Forbes et al. 1 metastasis; ovary HPD 5 PR 4
[57]
Dahlman et al. 1 CIS vulva HPD 3 CR —*
[58] 1 cervix, recurrence NR
Ha et al. [15] 3 cervix HPD 5 PR —
1 vaginal; papillary CR
Kennedy, [58] 1 endometrium HPD 2.5 PR 1 to 28 Both treated multiple
1 ovary Kodak light PR times for recurrence
Lele et al, [59] 21 (26) - All PHOTOFRIN{ 2 Pain at treatment site in
recurrent CR-7 all patients requiring
7 perineum, vulva, CR-2 analgesics
abdomen (skin) PR-2
11 vagina + cervix NR-7
Lobraico et al. 7 recurrent (45 PHOTOFRIN CR 34 3 1 second degree burn
[61] sites) {1.5 to 2.0 PR 8
Perianus NR 3
vagina
vulva
McCaughan et 2 primary, vagina HPD 2.3 0CR-2
al. [62] 1 vulva to 3 PR-1
2 metastatic CR-1, PR-1
Rettenmeier et Recurrent, vagina HPD 3.0 3 first degree burns
al. [63] 1 endometrium CR-2
3 cervix PR-3
1 vagina NR-1
1 Bertholin's gland
3 Intraepithelial HPD 5% PR-1 -Photosensitive
neoplasia NR-1
No FU-1
CR PR NR
Soma and 5 vagina HPD 2.5 4 1 0
Nutahara
[63] 5 CIS cervix 3 2 0 8 to 24
3 metastatic; 2 0 0
vagina
Ward et al 5 vagina HPD 5 CR-2 0.2 to 12
[64] recurrent PR-3 (mean 4.5)
CIS = carcinoma in situ NR = no response

CR = complete response FU = follow-up
PR = partial response
* Data not provided.
Table 10.4 shows results of a total of 100 women who have received PDT with PHOTOFRIN for
treatment of gynecologic cancers. Of those patients for whom a response is given, a complete response
was reported in 26 patients, partial response in another 24 patients, and no response in four patients;
four patients were not available for subsequent analysis. The duration of follow-up varied considerably
between studies. Edema, erythema, and necrosis accompanied by various levels of pain occurred in treated
sites 12 to 48 hours post-treatment.
PDT appears effective for the treatment of gynecological cancer, and is capable of achieving complete
eradication of visible tumors in patients with superficial disease. Improvements in and standardization
of light delivery and dosimetry to irregularly shaped anatomic areas such as the vagina, vulva and areas
of junction between cervical and vaginal mucosa are required for optimization of this form of therapy.
10.6 PHOTOFRIN PDT in Head and Neck Cancer
More than 40,000 cases of head and neck cancer (excluding skin cancer) are diagnosed annually in the
United States. Surgery and radiation therapy are the only curative treatments for cancer arising in the
head and neck. Cancers involving the lip, oral cavity (mouth, tongue, cheek), pharynx and larynx are
often treated by radical surgery with reconstruction wherever possible. In such patients, recurrence is
often followed by additional mutilating surgery, or surgery may not be possible. Radiation therapy is
also often given following surgical excision of tumor and, as maximal radiation therapy is often given
initially, follow-up radiation cannot be given on recurrence.
Data are shown in Table 10.5 for 198 patients treated with PDT with PHOTOFRIN or HPD for
head and neck cancer.66–78 Patients who had a mixture of primary, recurrent, and metastatic lesions
were included, with the histologic type of cancer unspecified in four reports. Cancerous lesions
involved most structures of the head and neck region. Patients were treated with HPD 2.0 to 5.0
mg/kg, or PHOTOFRIN 1.5 to 2.0 mg/kg. Many patients were retreated with PDT if the response
was inadequate after the initial treatment, and two to four treatments were used in each of three
patients in one study.68
In most studies, complete response was defined as absence of tumor; partial response was defined as
a reduction in tumor mass; and no response was defined as stable disease, disease progression, or patients
not available for analysis. Follow-up of response was continued for up to 34 months; however, most
patients were followed for less than one year after PDT.
Grossweiner et al.69 reported PDT treatment with PHOTOFRIN of head and neck squamous cell
cancers in nine patients (11 sites) with primary and one (one site) with recurrent, advanced disease.
Laser light treatment was delivered 24 hours following PHOTOFRIN injection. This paper is unique
in that theoretical dosimetry calculations were attempted using dimensions to account for the
irregularity of the tumor surface, and compared with the actual light fluence delivered to the sites.
A CR was obtained in 10 sites; the single partial response was obtained in a patient with an advanced
stage tumor that was thicker and “relatively inaccessible” to PDT in the nasopharynx. The site
reported as no response had been treated with lidocaine containing epinephrine (a vasoconstrictor)
just prior to treatment with laser light, which might have blocked oxygen delivery to the tumor.
Optimal light delivery consisted of a combination of surface illumination (using a microlens-tipped
fiber) and interstitial treatment with cylindrical diffuser insertion. Surface illumination was given
first, as interstitial cylindrical diffuser placement can cause bleeding that can affect absorbed light
energy. A 1 mm change in maximum tumor depth was found to have a significant effect on calculated
light dose. Dosimetry calculations thus varied up to a factor of 3 with respect to actual light fluence
delivered to tumor tissue.
A recent review of PDT for the treatment of oral, laryngeal, and head and neck cancers analyzed
results obtained for > 600 patients.80 Twenty-five patients with CIS and T1 squamous cell carcinomas
of the true vocal cord treated with PDT for cure obtained a CR after a single PDT treatment, and
the CR rate to a 79-month follow-up period remains 95%. A review of 217 patients with early
squamous cell carcinomas of the head and neck treated with PDT in the literature demonstrated an
89.5% CR rate.
The varying geometries and difficulty with determining true dosimetry in these cases render gen-
eralization difficult. However, it appears that the role of PDT in head and neck cancer may lie in
eradicating the superficial, early recurrence and “condemned mucosal” (pre-cancerous appearing
tissue) appearance of this disease, and thus this treatment modality has the potential to save patients
TABLE 10.5 Summary of Published Results with PDT in Head and Neck Cancer
Patients Drug Dose
Reference (sites) (mg/kg) CR PR NR Response Comments
Cai et al. [67] 26 primary or HPD 5 11 8 7 Follow-up to 34 months;

recurrent better responses in
untreated patients
Keller et al. [39] 2 adenoid HPD 3 2 1 0 Follow-up
3 early HPD 2-3 or 4 6 1 Follow-up to 18 months;
8 advanced PHOTOFRIN{ 1.5-2 all early stage had CR
Schuller et al. [70] 9 recurrent HPD 3-5 2 2 17 1 burn; 3 ulcer or
13 metastatic necrosis; 1 fatal
hemorrhage; follow-up
to 16 months
Taketa and 6 squamous HPD 3 1 5 0 2 laryngectomy; 3 with
Imakiire [71] T1 to T3 tumor; follow-up <(29
squamous)
31 primary HPD 5 and 2.5 15 16 0 HPD combined with
radiation
and recurrent provided enhanced
response
Zhao et al. [74] 50 Lip HPD 5 50 — — Only 4 recurrences; 4
developed
nodal metastases
Schweitzer [75] 8 PHOTOFRIN{ 2 4 3 1 CRs duration 6 Mos.
to 48 Mos.
Schweitzer and 5 oral PHOTOFRIN{ 2 3 2 — Duration of CRs 3-7
Visscher [76] months.
Kaposi's sarcoma
Freche and de 32 vocal cord HPD or 25 — 7 Duration of response 12
Corbierre [76] to
PHOTOFRIN{ 48 months
Gluckman [78] 8 advanced, HPD 3 or — 8 —
recurrent PHOTOFRIN{ 2
13 early Ca of PHOTOFRIN{ 2 11 2 — Duration of response 8-
40 Mos.
oral cavity and
oropharynx Duration of response 22
and 36 months
2 vocal cord PHOTOFRIN{ 2 2 — —
8 “condemned 7 1 — Duration of response 12
mucosa” to 48 Mos.
PHOTOFRIN{ 2
Wenig et al. [79] 26 early disease PHOTOFRIN{ 2 20 5 1 Duration of response
(various sites) 5-51 Mos.
CR = complete response
PR = partial response
NR = no response
from additional mutilating surgical procedures or to effectively treat patients with early disease who
are not eligible for or who refuse such surgery. Standardization of light treatment, and particularly
dosimetric methods, is necessary prior to the development of controlled clinical trials. It is possible
that combined light delivery and fluence measurement devices may eventually be incorporated into a
single device to simplify dosimetry in situ. PDT may also be of benefit as an adjuvant intraoperative
treatment of Stages III and IV tumors of the head and neck in conjunction with surgery and radiation
therapy to improve cure rates.
10.7 PHOTOFRIN PDT in Gastrointestinal Cancer
10.7.1 PHOTOFRIN PDT for Esophageal Cancer

Approximately 10,000 cases of esophageal cancer are diagnosed annually in the United States. While early
stage cancer (superficial disease without spread to lymph nodes) may be cured by surgery, 50% of
esophageal cancer patients are advanced to the point of inoperability at the time of diagnosis. Difficulty
swallowing because of obstruction of the esophagus by cancer (malignant dysphagia) is often seen as an
initial symptom of esophageal cancer. Although surgery may relieve malignant dysphagia, its use in
patients with advanced disease may not be justified because of high postoperative mortality rates (10 to
20%). Palliation of malignant dysphagia may be achieved using radiation therapy, intraluminal placement
of a tubular prosthesis, or removal of obstructing tumor endoscopically using Nd:YAG thermal ablation
therapy (as described above in the section on endobronchial lung cancer).
Patients who have been treated with PDT using HPD or PHOTOFRIN were of two types; those with
superficial or early esophageal cancer for whom surgery was not indicated82–85 and those with malignant
dysphagia due to advanced cancer and seeking palliation.86–90
Seventy-one patients with superficial (early) esophageal cancer were treated with PDT using HPD or
PHOTOFRIN.80–83 Complete responses were observed in 44 of 71 (62%) patients. Patients were treated
using cleaved fibers, microlens or cylindrical diffuser-tipped fiber optics, with side effects including
cutaneous photosensitivity. One group85 developed a new light delivery device for the treatment of early
esophageal cancer lesions.
Monnier et al.85 reported on the PDT treatment of 15 patients with superficial esophageal cancer using
HPD at 3 mg/kg or PHOTOFRIN at 2 mg/kg. PDT was carried out 72 hours after injection using a
special irradiator attached proximally to a Savary-Gillard dilator; 630 nm laser light dose varied from
60–150 J/cm2 (Figure 10.4). In this report, necrosis of esophageal areas not involved with tumor is
reported. While the use of the device described in Figure 10.4 allows dilatation of a normally collapsed
esophageal lumen and presumably uniform illumination, the use of semicircular mirrors to allow 180-
degree rather than 360-degree illumination may have increased fluence and thus total light dose beyond
that desired. It is also possible that even mild stretching of the esophageal wall by an inelastic device may
serve to compress blood vessels and compromise blood flow slightly, which could increase the sensitivity
of all tissues to the photodynamic effect. Studies that have used bare cylinder diffuser-tipped fibers
(Figure 10.1) or a cleaved fiber placed within a thin-walled balloon (see below) have not reported
nonspecific necrosis. Complete responses were observed in 15 of 15 cases (100%), but local recurrence
was observed within 1–3 months in three patients. The remaining patients were locally tumor-free for
from 9–60 months.
PDT has been used for the treatment of malignant dysphagia in uncontrolled studies on 55 patients.86–90
Relief of dysphagia has been recorded in 52 of 55 (95%) treated patients. Complications included
cutaneous photosensitivity (including painless tanning), and esophageal scarring resulting in strictures
(narrowing) managed by mechanical dilatation. Two patients with esophageal obstruction so advanced
that a guide wire could not be passed, which rendered Nd:YAG laser treatment inadvisable, were treated
with PDT and obtained resolution of their malignant dysphagia, which had been so advanced they were
unable to swallow their own saliva.90
Thus, PDT appears to have efficacy both in the treatment of early esophageal cancer and as a method
of palliation for malignant dysphagia. PHOTOFRIN has been approved for the treatment of patients
with completely obstructing esophageal lesions using data from 17 patients provided by a Phase II single-
arm protocol for treatment.90 Following a single course of therapy, 94% of patients obtained an objective
tumor response, and 76% of the patients experienced at least some palliation of their malignant dysph-
agia.53 Before treatment, these patients, on average, had difficulty swallowing liquids, even saliva. The
median duration of clinically meaningful benefit to these patients was 69+ days.
For the treatment of long esophageal tumors, the development of flexible but strong cylinder diffusers
of 5 to 10 cm length would allow treatment of long tumors in a short time. Such long cylinder diffusers
Removable PTFE diffusing

tube (see inset)
Optical Graduated Savary-Gillard
Fiber end
fiber tube dilator
Pin photodiode
X-ray marker
Chromel-const.
thermocouple Ag thermal contact Al mirrors Positioning spring
PMMA bulb
FIGURE 10.4 Line drawing of light-diffusing cylinder developed by Monnier et al.85 for the delivery of light to early
cancers of the pharynx and esophagus. Reprinted with permission of the publisher, Lasers in Medical Science.
would have to maintain homogeneity of light dispersion along their lengths. High-output lasers would
be needed for such treatments because, with the current recommended power density of 400 mw/cm
fiber, lasers would have to deliver a stable output of 2 to 4 watts at the fiber tip.
PHOTOFRIN has also been used to treat the precancerous condition of Barrett’s esophagus with high-
grade dysplasia. Barrett’s esophagus is an acquired condition in which the normal squamous epithelium
of the lower esophagus becomes replaced with a columnar epithelium with intestinal metaplasia.92 It is
named after Norman Barrett who, in 1950, described a case of columnar epithelium in a congenitally
short esophagus.93 The definition has been refined over the years, but its significance arises from its
potential for malignant transformation.
Adenocarcinoma of the esophagus is increasing in incidence more rapidly than any other cancer in
the Western World.94 The major risk factor is Barrett’s metaplasia, which is thought to arise as a healing
mechanism in response to chronic injury from gastroesophageal reflux (GERD).95,96 In a proportion of
cases, this metaplastic tissue can progress to dysplasia and malignancy.97,98
Barrett’s esophagus is a relatively common condition, with an estimated incidence of 0.4% of the
general population99 and 12% in patients with chronic gastroesophageal reflux.100 The metaplastic epi-
thelium confers a lifetime risk of 10% of developing adenocarcinoma,101 which equates to one in 52 to
one in 441 patient years, or 30–125 times the risk of the general population.98,102–104 Furthermore, this
risk increases significantly with increasingly severe dysplasia.102,105 Overholt et al. have published the
largest series of patients treated with PDT.106 They have treated 100 patients, 87 of whom had dysplasic
Barrett’s esophagus. They were all treated with PHOTOFRIN and red light (630nm) and followed up for
a mean of 19 months (range 4–84 months). The results were encouraging, with 43% having eradication
of the entire Barrett’s segment, and there being a 75–80% replacement with squamous epithelium in the
rest. Dysplasia was eliminated in 78 patients, although two developed high-grade dysplasia underlying
the neo-squamous epithelium. Significant oesophageal strictures that required formal dilatation devel-
oped in 34% of patients. In almost 20% of cases over the period of follow-up, there was recurrence of
dysplasia that required further treatment with PDT.
10.7.2 PHOTOFRIN PDT for Colorectal Cancer

Approximately 150,000 cases of colorectal cancer are diagnosed in the United States each year, represent-
ing 15% of all cancers. Surgery is the only potentially curative form of treatment, with degree of spread
found at the time of surgery (i.e., limited to within the colon, spread to adjacent lymph nodes, or spread
beyond the colon or rectum) predictive of the risk of recurrence. Chemotherapy following removal of
all visible tumor, (a procedure termed adjuvant chemotherapy) with 5-fluorouracil and levamisole is
currently recommended as a means of increasing the time to recurrence of disease.
Results have been obtained that show accumulation of HPD in colon cancer,89 and which suggest that
PDT may be superior to thermal ablation therapy for this class of tumors in light of the resistance of the
colon to perforation despite over-treatment with light and HPD combinations.90 Exposure of normal rat
colon to PDT at light- and drug-dose combinations sufficient to achieve full thickness necrosis had no
effect on the mean bursting pressure of the colon even 96 hours post-PDT. Thermal necrosis of compa-
rable extent is associated with a 22% perforation rate and weakening of the bowel wall. The authors
postulate that lack of mechanical damage to submucosal collagen allows retention of mechanical strength;
healing was by regeneration and was complete 2 weeks after treatment.
Barr et al.108 reported on a pilot study of PDT of 10 patients with colorectal cancer using HPD (2.5
mg/kg) as photosensitizer. All patients presented with symptoms (bleeding, overflow diarrhea, tenesmus,
or discharge). All tumors were 5 cm or less in length, and were treated with 630 nm laser light 48 hours
after HPD injection using an interstitial method with a cleaved fiber optic with the cladding removed
from the distal 1mm. Power at the fiber tip was 50-l00mW to avoid any thermal damage, and a total
light dose of 50 J was given with multiple doses “such that light fields just overlapped” to treat the entire
tumor. The authors conclude that PDT has promise in the treatment of small, or residual, tumors of the
gastrointestinal tract and that a combination of Nd:YAG laser (for tumor debulking) and PDT might
produce optimal results.
While the data on HPD localization91 suggests that adenomas as well as adenocarcinomas can be
effectively treated with PDT, it is apparent that the use of a cleaved fiber provides suboptimal light
distribution. Future work should, if possible, use cylinder diffuser-tipped fiber optics, which can provide
light to sufficient depths to rapidly treat such tumors. Large, sessile adenomatous polyps or villous
adenomas may prove to be effective targets for PDT treatment as well.
10.7.3 PHOTOFRIN PDT for Early Gastric Cancer

The Japanese tradition of screening for gastric cancer has allowed the PDT treatment of patients who
refuse surgery or for whom surgery is contraindicated. All of the 53 patients thus treated with HPD and
cleaved-fiber endoscopic laser light delivery had Type II cancers, defined as ulcerative and circum-
scribed.109–113 Thirty-two patients were treated with PDT alone, and of these, 28 (82%) had no remaining
tumors visible by endoscopy or biopsy. Partial responses in this group tended to occur in patients with
tumors greater than 4 cm in diameter. Epigastric pain following PDT appears to be the only complication
of treatment. PHOTOFRIN has been approved and is marketed in Japan for this indication.
While treatment of early gastric cancer has great potential for treatment with PDT, several technical
problems may need to be overcome to allow optimal treatment. The shape of the stomach, ongoing
peristalsis and, perhaps most important, deep gastric mucosal folds (rugae) which may prevent even
light distribution, must all be taken into account. It may be possible to use some device to smooth the
mucosal folds while providing light illumination through a diffusing medium, similar to that described
by Muller and Wilson for the treatment of brain tumor cavities.114
10.8 PHOTOFRIN PDT for Intraoperative Abdominal

or Thoracic PDT
In the evolution of clinical PDT, one of the more promising areas appears to be in the field of intraop-
erative PDT, in which PDT is used as an adjunct to surgical resection. Nambisan et al.115 pioneered in
this area. Ten patients with recurrent retroperitoneal sarcomas were injected intravenously with either
HPD (2.5–5 mg/kg) or PHOTOFRIN. If no extensive organ involvement was found during surgery, an
attempt was made to completely resect the tumor, and areas of fluorescence discovered using a UV lamp
were also resected. After resection, the field was illuminated with 630 nm laser light using a microlens-
tipped fiber optic to a total light dose of 30 to 288 J/cm2. In 8 out of 10 cases, complete surgical resection
was possible, making the treatment of these patients of adjuvant type. No complications or side effects
were noted except for photosensitivity. McCaughan et al.116 reported on the intraoperative therapy of
extrahepatic bile duct tumors in a 57-year-old female. Over the course of 4 years, the patient underwent
a total of seven treatments, with the last six administered via a U-tube tract. Median life expectancy for
this condition ranges from 5 to 11 months if curative resection is not performed. Although anecdotal,
this report indicates that interventional PDT may be a treatment option for cholangiocarcinoma.
The National Cancer Institute of the United States National Institutes of Health (NIH) has reported on a
Phase I study of PDT with PHOTOFRIN for disseminated intraperitoneal neoplasms.117 Patients (n = 23)
were injected with PHOTOFRIN prior to laparotomy and ,48 to 72 hours after injection, underwent
debulking to leave behind no neoplastic nodules > 5 mm in diameter. Following debulking, they were
treated with 630 nm laser light, using in situ dosimetry in which four sterile photodiodes were sewn into
the peritoneal cavity and a microcomputer was used to provide baseline reading of fluence and light
dose. Dilute lipid suspension was used (0.02–0.05% Intralipid) within the abdomen to enhance light
diffusion. The authors thought that the maximal tolerated dose of PDT to the entire peritoneal surface
had not been determined; the highest doses of drug and light reported used in the study were 2.5 mg
PHOTOFRIN/kg and 3.0 J/cm2 630 nm wavelength laser light.
At the National Cancer Institute, NIH, intraoperative PDT has also been extended to the treatment
of pleural malignancies.118 A Phase I study has been reported on the use of PDT for intrathoracic treatment
after thoracotomy with debulking of all gross tumor to 5 mm thickness. Eligible patients had disease
confined to one hemithorax. All patients received 2 mg/kg PHOTOFRIN and, 48 hours after injection,
thoracotomy was performed with resection of gross disease. Laser light dosimetry was performed using
seven photodiodes sewn in situ throughout the chest; 0.02% Intralipid was used as a light diffusion aid.
All three patients reported treated were discharged 7–10 days after surgery, and no complications were
reported. Lofgren et al.119 have reported on transthoracic endoscopic PDT as treatment for malignant
mesothelioma (tumor originating in the pleural lining of the lung) in a patient. A model of the pleural
cavity was used to determine light dosimetry; the patient was given PHOTOFRIN 2 mg/kg and 48 hours
after injection, thoracoscopy was performed. The entire surface of the pleural cavity (as could be reached
via thorascopy and fiber optics) was reportedly exposed to a total light dose of 20J/cm2 from a gold vapor
laser. The entire procedure took 6 hours under epidural anesthesia; in situ dosimetry was performed
using a probe inserted via a separate puncture in the thoracic wall. The patient was followed for 10
months without signs of tumor progression; all laboratory values returned to normal.
Intraoperative PDT may, in the future, become a reality for primary tumor treatment. At the present
time, limitations on the treatment relate to the large areas (particularly for intraperitoneal therapy) that
require treatment with light. Intraperitoneal therapy may require delivery of light to a total area of up
to 20,000 cm2. Laser power is therefore, at the present time, a limiting variable in the expansion of this
indication of PDT. However, the above mentioned work suggests that adjuvant intraoperative therapy of
tumors at risk of recurrence, as well as methods to convert surgical partial responses to complete tumor
responses (and therefore “cures”) should be the goals of intraoperative PDT. Development of longer-
wavelength photosensitizers as well as alternative methods of activation of photosensitizers should be
explored in order to minimize the time necessary for the surgical PDT procedure.
10.9 PHOTOFRIN for Intraoperative PDT in the Treatment

of Intracranial Tumors
Primary intracranial tumors (which arise within the central nervous system), the majority of which occur
in persons over the age of 45, represent a small percent of total cancer incidence (approximately 8,000
cases annually). Malignant intracranial tumors are generally removed only by surgery. Patient prognosis
and survival depend greatly upon the type of tumor and how much can be removed by surgical excision.
Tumors of minimal malignant potential are often encapsulated and easily removed in toto. The most
malignant and aggressive of intracranial tumors is glioblastoma multiforme, representing up to 9% of
all malignant intracranial tumors found in persons over the age of 45. Glioblastoma is not encapsulated,
and individual tumor cells indistinguishable among normal brain tissue at the time of surgery may spread
several centimeters away from the tumor bed. Despite surgery and postoperative radiation therapy (the
standard method of treatment), median survival remains approximately 1 year following diagnosis. Upon
recurrence, survival is only 4 to 6 months. PDT has been examined for its ability to treat and prevent
recurrence in glioblastoma and non-glioblastoma tumors in man.120–127
To laser
Protective tubing
Fiber holder
Tightening screw
O - ring
Out In
1 cm
Retaining flange
Optical fiber
Rubber 'balloon'
Nutralipid
FIGURE 10.5 Line drawing of the balloon device developed by Muller and Wilson for delivery of light to the
tumor bed left following craniotomy and tumor resection.107 Reprinted with permission of the publisher, Lasers
in Medical Science.
Kaye and Hill120 reviewed progress in this area.121–127 The first 10 patients treated with PDT for intracranial
tumors were reported 21 years ago.122,123 Laser light was delivered via fiber optic probes inserted directly
into the tumor or cyst cavity intraoperatively, but total light doses did not exceed 10 J/cm2.
A new device for delivery of light to post-resection tumor beds was developed by Muller and Wilson,
who first reported on its use in the treatment of 32 patients with malignant supratentorial gliomas by
intraoperative PDT.114 These authors have reported on a series of 50 patients.125 Eighteen to 24 hours
following photosensitizer injection, craniotomy (opening of the skull) with maximal tumor resection
was performed followed by photoradiation of the resultant cavity with an inflatable balloon applicator
filled with 0.1% lipid emulsion to scatter the light produced by a cleaved optical fiber inserted into the
balloon (Figure 10.5). Patients were treated with laser light doses of 8–68 J/cm2. Complete responses
(disappearance of residual tumor by computed tomographic scans) were observed in 12 of 45 primary-
tumor patients (19%); four of these patients had recurrent tumors. The authors concluded that the “PDT-
associated response rate” was 35%.
There were no significant differences in median survival for patients with de novo tumors as compared
with patients who had recurrent tumors. While this series is too small and heterogeneous with respect
to pathology and primary or recurrent disease to draw conclusions, it is interesting to note that there
was a significant difference in survival between patients receiving high (>1500 J total) and low (<1500
J total) light doses in the nonglioblastoma multiforme group. Those patients receiving the high-dose
light had a median survival of 18 months (n = 11) compared with 6.6 months for those patients receiving
the low-dose light (n = 11). Using an intrinsic light detection device, Muller and Wilson114,121,125 deter-
mined the light penetration depth to be 2.9 mm in tumor (1.2–4.5 mm) and 1.5 mm in normal brain.
Kaye and Hill120 reviewed their experience on the treatment of 45 patients with intraoperative PDT,
44 of whom had high-grade gliomas (15 recurrent glioblastoma). All patients received HPD (5 mg/kg)
and 24 hours later underwent craniotomy with radical tumor excision followed by PDT using a flat-cut
fiber for light delivery of up to 240 J/cm2 to the tumor bed as “adjuvant therapy,” although residual tumor
was left at the excision site in 19 patients. Laser light was produced by an argon-pumped dye laser for
the treatment of nine patients, and by gold-vapor laser for 36 patients. If a cavity resulted from tumor
resection, it was filled with 0.5% (v/v) Intralipid to scatter light evenly throughout the cavity. Total light
dose varied from 70–240 J/cm2; the temperature of the tumor bed was kept at 37°C by frequent irrigation.
Patients in this series were followed up to 39 months, and survival curves in various groups were
compared. As in the studies of Muller and Wilson,125 PDT was found to have no significant impact on
survival time in patients with recurrent tumors (median survival time 6.8 months). However, median
survival of patients with primary tumors treated with resection and PDT followed by postoperative
radiotherapy (45 Gy in 20 divided doses) had not yet been reached at the 39-month follow-up point. It
is also interesting to note that , in this series,103 patients who received < 120 J/cm2 had a median survival
of approximately 12 months, while those who received >120 J/cm2 had not yet reached median survival
at the 39-month follow-up period. However, numbers of patients in the low- and high-power-treatment
groups were not provided, nor is it known how many had primary or recurrent disease. No postoperative
complications or evidence of increased intracranial pressure were observed. In a follow-up study121 of 56
patients with recurrent tumors, all of whom had failed radiation therapy, the mean survival time for
patients receiving PDT with PHOTOFRIN for glioblastoma, malignant astrocytoma, and mixed astro-
cytoma-oligodendroglioma was 30, 44, and > 61 weeks, respectively. For patients undergoing surgery
alone, median survival was only 20 weeks. The survival of patients with malignant astrocytoma was again
related to light dose, with those receiving a total of > 1800 J. surviving longer (64 weeks median) than
patients who received < 1800 J (27 weeks median survival).
Powers et al.127 reported on the stereotaxic placement of cylindrical diffuser-tipped fibers (Figure 10.1)
for the treatment of recurrent inoperable malignant brain tumors with PDT using PHOTOFRIN . Six
patients had recurrent supratentorial malignant gliomas (four anaplastic (undifferentiated)) astrocyto-
mas, one glioblastoma and one gliosarcoma and one patient had an intracranial metastatic melanoma.
Patients received 2 mg/kg PHOTOFRIN intravenously 24 hours prior to laser light exposure at 630 nm
wavelength. Cylindrical diffuser length was chosen to closely match axial length through the epicenter
of the tumor. A power of 200 mw/cm of diffuser was delivered to a total light dose of 400 J/cm diffuser
tip. The tumor response to PDT after light treatment was estimated by pre- and post-PDT computed
tomography (CT) and magnetic resonance imaging (MRI) scans; 31P MR spectroscopy was used to
determine metabolic changes in tumor tissue. Following PDT, the solid tumor volume defined by
radiographic appearance, changed for all six gliomas from a homogeneously enhancing to a centrally
non-enhancing lesion with a greater total volume than that originally defined by contrast enhancement
in the preoperative CT images. The patient with metastatic melanoma showed no CT or clinical evidence
of tumor response. Two patients, both with anaplastic astrocytomas, were stable without evidence of
active disease at 45 and 35 weeks post-PDT; radiologic evidence of recurrence was seen in the other five
patients, generally within 6 to 8 weeks of treatment. Glioma regrowth occurred only outside the contrast-
enhancing rim noted on post-PDT CT scans. It is clear from this study127 that intratumoral PDT produces
tumor necrosis but that additional treatment of the surrounding tissue is required to destroy infiltrating
cells presumably responsible for tumor recurrence.
The results from the above studies can only be described as preliminary and suggestive, particularly
with respect to differences in survival between groups of patients receiving “high” and “low” light
doses.120,125 Nearly all of the published reports have suffered from lack of stratification of patients on the
basis of primary versus recurrent tumors, mixing of patients with different kinds of intracranial tumors,
and lack of uniformity in light delivery and dosimetry methods. Despite the fact that Muller and Wilson125
measured the penetration depth of light into brain tumor as 2.9 ± 1.5 mm, they and Powers et al.127 note
that the depth of tumor necrosis may be several times the apparent light penetration depth. The reason
for this discrepancy is not clear at the moment, but may relate to photobleaching, vascular occlusion and
local inflammatory or cytokine-mediated effects. It is clear that new light-delivery devices must be
developed to adequately treat large areas of the brain to depths of several centimeters. The balloon device
described by Muller and Wilson114 has been criticized as inappropriate for uneven cavities or for resections
resulting in a flat surface following tumor resection.120
Additional clinical trials need to be performed to determine the potential of PDT in the treatment of
brain tumors. Perria et al.128 have proposed that all patients with primary brain tumors should be admitted
into such a study at first presentation. Patients would receive surgical tumor excision and PDT followed
by radiation therapy, and would be compared with patients undergoing standard resection followed by
radiotherapy. A randomized trial comparing the two methods of treatment would be the ideal (and only)
method of determining the potential contribution of PDT to the other modalities used in neurosurgical
oncology. Such multicenter trials must use uniform methods of light delivery and light dose escalation
to a uniform tumor type. The findings of Powers et al.127 should stimulate the initiation of additional
trials utilizing stereotaxic placement of fiber optics for treatment of unresectable tumors to fully explore
the use of PDT in this indication. The potential of steroid use given after administration of laser light to
minimize cerebral edema effects should also be investigated.
10.10 PHOTOFRIN PDT in the Treatment of Ocular Cancer

Malignant melanoma accounts for approximately 70% of all eye (ocular) malignancies, and occurs at an
incidence approximately 12% that of skin melanomas (about 20,000 cases per year, but varying with
geographic location). Initially, therapy for ocular melanomas consisted of enucleation (surgical removal
of the globe of the eye in its entirety). More recently, however, attempts have been made to use radiation
therapy, brachytherapy and laser photocoagulation for selective destruction of the tumor and preservation
of the eye. The equivalence of newer methods of treatment with surgery, however, remains to be proven.
PDT has been used for the treatment of ocular melanoma.129–133
Bruce and McCaughan133 treated the largest series, 24 patients, with uveal melanoma over a 2-year
period and provided follow-up for as long as 7 years. HPD was administered to all patients at 2.5 mg/kg
body weight. Light produced by an argon-pumped dye laser at 630 nm wavelength was delivered via the
transpupillary route as well as directly over the base of the tumors. Total light doses ranged from 300 to
3000 J/cm2. Complications included transient initially reduced vision, cataract, exudative retinal detach-
ment and neovascular glaucoma as well as photosensitivity. Tumors were divided into three groups based
on size; small (< 500 mm3); medium (500–1000 mm3) and large (> 1000 mm3). All groups responded
well initially with avascular necrosis. In the small and medium-sized tumor groups, the tumor mass was
completely replaced by scar tissue. In the large-tumor group, tumor destruction was incomplete, despite
additional PDT treatments, and enucleation was required in each case. During 7 years of follow-up, six
of the 24 patients died, with three deaths due to metastatic melanoma. Ten patients underwent enucleation
for recurrence of tumor at the edge of the treated area or regrowth after a partial response.
Thus, PDT appears to have good promise for the treatment of ocular melanomas of ≤ 1000 mm3
volume.133 It has been suggested that an alternate method of ocular melanoma treatment involve treat-
ment of the choroid surrounding the tumor to close off the choroidal blood supply, rather than concen-
trating on direct light treatment of the tumor itself.129 Although the collective data suggest that high light
doses (in excess of 1000 or 2000 J/cm2), might be optimal for treatment of melanoma, clinical trials for
dose-ranging studies are required to test this possibility. The fact that melanoma tends to be a heavily
pigmented tissue and thus attenuates light rapidly over a short distance, is supported by the complete
response seen in amelanotic melanoma at a light dose that produced only partial responses, at best, in
pigmented melanomas.132 Development of devices specific for delivery of light to ocular structures may
be able to increase the complete response rates seen for ocular melanoma.
Retinoblastoma is the most common primary ocular tumor in children, with an incidence of approx-
imately 200 per year. It arises from cells within the nuclear (outer) layer of the retina, and 80% of cases
are diagnosed in children less than 4 years old. Surgery (enucleation) or radiation therapy may be used
for treatment, although approximately 50% of patients receiving radiation therapy alone eventually
require enucleation. Patients with small, unilateral tumors may be treated with cryosurgery or laser
photocoagulation to preserve both the eye and vision. However, even patients with large tumors and
distant metastasis can have a 5-year survival approaching 90%, although the chance for complete disease
control is only about 30% in such cases.
In retinoblastoma, PDT appears to be able to delay time of enucleation but, in the studies reported,132,134
did not produce long-term responses. The vitreous cancer seedings, which receive nourishment not from
blood, but from vitreous humor, may not contain sufficient photosensitizer to allow a good response at
the light doses used in these trials, and might account for the poor results that were obtained. Future
trials may consider injection of small quantities of photosensitizer directly into the vitreous in the vicinity
of the tumor. The presence of lipophilic photosensitive dyes may allow selective uptake by the tumors
and improve the tumor response.
10.11 PHOTOFRIN PDT in the Treatment of Cutaneous

and Subcutaneous Tumors
The most common forms of skin (cutaneous) cancer are basal cell and squamous cell carcinoma, which
generally occur on sun-exposed areas such as the hands, neck, and head. More than 500,000 such skin
cancers are reported annually in the United States. Surgery is generally performed and is curative when
performed in early stages of either cancer. However, curettage, electrodessication and laser vaporization
can be used as alternative methods of therapy for superficial lesions. For the treatment of difficult and
recurrent skin cancers, Mohs’ micrographic surgery is used, a specialized technique in which serial
horizontal sections of excised tissue are mapped and examined by frozen section examination, with
excision continued until margins are microscopically free of tumor. Cryosurgery, topical 5-fluorouracil
and radiation therapy have also been used for selected patients with superficial skin cancer. In patients
with recurrent or aggressive skin cancer, PDT has been examined as a potential treatment modality as a
nonsurgical treatment on patients in whom surgery is not indicated because of poor health, and in
patients with skin cancers in sites where surgical excision might lead to scarring upon healing. Rare forms
of aggressive skin cancer that occur over many sites simultaneously (Bowen’s disease, basal cell nevus
syndrome) have also been treated experimentally with PDT due to the difficulty of adequate therapy
with existing procedures.
Cutaneous or subcutaneous cancer can also occur as a consequence of metastatic disease from other
sites. Local recurrence in the chest wall following mastectomy may occur in up to 27% of patients. Up
to 80% of these patients may have an initial response to radiation therapy, but up to two-thirds will
develop another local recurrence. Systemic chemotherapy and hormonal therapy have also been used to
treat these patients. PDT has been investigated as a potential therapy in these patients as well.
PDT has been used to effectively treat surface lesions, whether of primary skin cancer origin or
metastatic from a distant primary source. A summary of publications135–143 on the use of PHOTOFRIN
PDT in this indication is given in Table 10.6.
Schuh et al.140 reported on the treatment of 14 patients with breast cancer metastatic to chest wall with
PDT using PHOTOFRIN (1 or 2 mg/kg). All patients had been previously treated with multiple modalities.
It is perhaps important to note that light doses as high as 288 J/cm2 did not cause necrosis of normal
tissue. However, “in previously heavily irradiated areas” skin necrosis was observed to occur at light doses
TABLE 10.6 Summary of Published Studies Using PDT for Treatment of Cutaneous and Subcutaneous Tumors
Method of Results
Patients (sites) Dose of Light Follow-up
Reference Disease Drug (mg/kg) Delivery CR PR NR PD (months
Bandieramonte 7 (61) HPD S 26 16 7 3 (Evaluable)

et al.[117] (43 Basal cell) (3) 60-120 J/cm2
(18 Metastatic
breast cancer)
Gilson et al. 6 (34) PHOTOFRIN S 16 NA NA NA (3)
[118] Squamous cell, 1-2 mg/kg 25-100 J/cm2
lung cancer
McCaughan 27 HPD S 48 19 NA NA (12)
et al. [119] Basal cell (3) (20-30 J/cm2)
cancer (7) PHOTOFRIN 1
Squamous cell (2)
cancer (3)
Melanoma (3)
Metastatic
breast
cancer (12)
Pennington et 6 (53) HPD S 36 NA NA NA (12)
al. [120] Basal or (5) (30 J/cm2)
squamous
cell cancer
Robinson 2 (>500) PHOTOFRIN S >5 0 0 0 (6)
et al. [121] 00
Bowen's (2) (25 J/cm2)
Disease
Schuh et al. 14 PHOTOFRIN S 2 9 3 (6)
[122] Metastatic (1-2) (36-288 J/cm2)
breast
cancer 1
(350-440
J/cm2)
Tse et al. 3 (40) HPD S 33 7 4 0 (12-14)
[111] Basal cell nevus (3) (38-180 J/cm2
Syndrome
Waldow et al. 10 (155) HPD S 11 30 8 (8-24)
[123] 7
Metastatic (3) (8-60 J/cm2)
breast cancer PHOTOFRIN
Basal cell (2)
cancer
Bowen's
disease
Wilson et al. 37 PHOTOFRIN S 13 18 — — (Minimum
[124] 3 12)
(151) (1) (180-233
J/cm2)
Basal Cell
Cancer
Sperduto et al. 20 PHOTOFRIN S 4 9 7 — (1-18)
[125] {
Metastatic (1.5) (20-359 J/cm2)
breast cancer
S-Surface; I-Interstitial; NA-Not Available; CR-Complete Response; PR-Partial Response; NR-No Response; PD-Progressive
Disease
in the 104–144 J/cm2 range. No attempt was made to correlate degree of skin photosensitivity with extent
or dose of prior radiation therapy.
It is apparent that large numbers of surface lesions can be treated repeatedly with PDT to get a desired
effect. Robinson et al.139 described the treatment of two patients who together had > 500 lesions of
Bowen’s disease (intraepidermal carcinoma). Up to a total of five treatment sessions were given, resulting
in complete clearing of the lesions by the last 6-month observation period post-treatment. The authors
noted that halving the dose of PHOTOFRIN for one treatment session did not diminish the photosen-
sitivity reactions but “significantly reduced the efficacy of treatment.” Tse et al.129 treated multiple lesions
of the basal cell nevus syndrome in three patients. Nevoid basal cell carcinoma syndrome (NBCS) is a
rare inherited autosomal-dominant disease. Nevoid basal cell carcinomas are multiple, have an early age
of onset, and are indistinguishable microscopically from basal cell carcinoma. Of the 40 lesions treated,
33 had complete responses and seven partial responses (CR + PR = 100%). CRs were followed for 12–14
months, and four recurrences (10.7%) were observed.
The National Cancer Institute experience using PDT with PHOTOFRIN for the treatment of 20 patients
with locally recurrent (chest wall) breast carcinoma has been described.143 Complications included pain,
bruising, blistering, ulceration and necrosis in areas of tumor involvement. It is to be noted that healing
was observed in all cases. The authors conclude that the depth of tumor involvement in chest wall
metastases from breast cancer generally exceeds the penetration of 630 nm wavelength light applied by
surface treatment, and that this may account for the short periods of response as well as the low numbers
of patients responding. Responses did not show a clear relationship to power density or to total light
dose. No attempts were made to deliver therapy via interstitial fiber placement in this study.
Thus, it appears that PDT has the potential of becoming a preferred method for the treatment of
certain cutaneous and subcutaneous cancers. In addition to efficacy at removing lesions, the healing
process of lesions occurring as the result of PDT treatment (which may take from 2 to 12 weeks depending
on the size and depth of the necrosis), results in excellent cosmetic results often mentioned as “free of
scar formation.”136 Cutaneous photosensitivity and pain during and after treatment at the site of treatment
are generally mentioned side effects. For PDT to reach its potential in this indication, standardization of
drug and light dose through the use of multicenter trials will be necessary. Standardization of the method
of light delivery, particularly if interstitial light delivery is to be used, must also be carried out. The
contribution of prior radiation therapy to normal skin necrosis at usual therapeutic doses of light and
drug must also be determined if treatment of cutaneous breast cancer metastases is to become a standard
indication for PDT treatment.
10.12 New Photosensitizers for PDT

PHOTOFRIN, although the first approved photosensitizer for use in PDT, has characteristics that
could be improved upon for greater acceptance in certain clinical indications. With PHOTOFRIN, a
waiting period of 40 to 48 hours has been recommended to allow the photosensitizer to “wash out”
of normal tissue to avoid a lack of selectivity in tissue destruction when laser light is applied.3
Cutaneous photosensitivity may persist for 4 to 6 weeks or even longer, limiting the mobility of the
treated individual to periods during which the sun is down or cloudy weather predominates (unless
complete covering up in protective garments is carried out.2 PHOTOFRIN is also a complex molecular
mixture, containing thousands of species of oligomers, thus rendering it extremely difficult to deter-
mine which species are responsible for antitumor efficacy or cutaneous photosensitivity. Photosen-
sitizing agents, which are single molecular entities, provide brief periods of cutaneous
photosensitivity, and allow treatment of patients with light shortly after administration (preferably
on the same day as photosensitizer dosing), have been sought. The following is a brief discussion of
individual compounds that have been approved for treatment of various indications and represent
the second generation of PDT photosensitizers.
10.12.1 VISUDYNE™ (Verteporfin)

VISUDYNE (QLT, Inc.) is a chlorin produced, as is PHOTOFRIN, from hemoglobin as starting material.
It is a chlorin with the longest wavelength absorbance peak at 690 nm and is more hydrophobic than
PHOTOFRIN, necessitating formulation in organic solvent or liposomes.144 Peak tissue levels of VISU-
DYNE are achieved 3 hours following intravenous injection in rodents, and the compound appears to
“wash out” of normal skin 3 or 4 days following injection. It is hoped that the 690 nm wavelength of
activation will allow deeper tissue penetration for greater efficacy in bulky tumors than is seen with
PHOTOFRIN, which uses 630 nm wavelength light for activation of the PDT process. It is believed that
VISUDYNE PDT mechanisms of tumor necrosis are similar to those of PHOTOFRIN and involve damage
to tumor vasculature. A phase I study in patients with primary skin cancer or cutaneous metastases of
cancer from other primary sites145 reported that patients were first given an intravenous infusion of
0.2–0.5 mg/kg of VISUDYNE in a liposomal formulation. The tumors were then irradiated with 50–150
J/cm2 of 690 nm light from an argon-pumped tunable dye laser via front surface illumination 3 to 4
hours after VISUDYNEl injection. Of 64 skin tumors from the first 15 patients, the overall (complete +
partial) tumor response was 74%, with higher response rates observed using high dose ranges of either
drug or light. Side effects of tumor and normal skin treatment (normal skin was included in the field of
illumination) included edema and erythema of normal skin.
10.12.1.1 VISUDYNE PDT for the Treatment of Age-Related Macular Degeneration
Age-related macular degeneration (AMD), a deterioration of the central portion of the retina, is the
major cause of severe, irreversible vision loss and blindness in the United States and other developed
countries (see Ref. 146 for review). There are two forms — the atrophic and the neovascular, exudative.
The neovascular, exudative form includes serous or hemorrhagic detachment of retinal pigment epithe-
lium and choroidal neovascularization, which lead to leakage and fibrovascular scarring. VISUDYNE
PDT received marketing approval in the United States and other countries during 2000 for the treatment
of AMD in patients with predominantly classic subfoveal choroidal neovascularization. For PDT of AMD,
VISUDYNE is injected intravenously to a total dose of 6 mg/m2 and 689 nm laser light is delivered at an
intensity of 600 mW/cm2 over 83 sec to provide a recommended light dose of 50 J/cm2.147 The treatment
spot size was 1000 microns larger than the greatest linear dimension of the lesion on the retina to allow
a 500-micron border and to ensure full coverage of the lesion. The phase III trials were double-masked,
placebo-controlled, randomized studies enrolling a total of 609 patients (VISUDYNE 402, placebo 207).
During these studies, retreatment was allowed every 3 months if fluorescein angiograms showed any
recurrence or persistence of leakage. The placebo control consisted of intravenous administration of
Dextrose 5% in water, followed by light application identical to that used for VISUDYNE PDT. A planned
analysis of safety and efficacy conducted at 1 year statistically favored VISUDYNE for the visual acuity
endpoints. The subgroup of patients with predominantly classic choroidal neovascularization lesions was
more likely to exhibit a treatment benefit. For the primary efficacy endpoint (percentage of patients who
lost < 3 lines of visual acuity), these patients showed a difference of 28% between treatment groups (67%
for VISUDYNE PDT patients compared with 39% for placebo patients, P < 0.001). The most frequently
reported adverse events to VISUDYNE included headaches, injection site reactions (including extrava-
sation and rashes) and visual disturbances, including blurred vision, decreased visual acuity and visual
field defects. Severe vision decrease of four lines or more within 7 days of treatment was reported in
1–4% of patients.
10.12.2 LEVULAN® (Aminolevulinic Acid HCl, ALA) PDT

Levulan (DUSA Pharmaceuticals, Inc.) is itself not a photosensitizer, but a sort of prodrug, as it is the
natural metabolic precursor of the endogenous tissue photosensitizer protoporphyrin IX (PPIX).128 PPIX
is an immediate biosynthetic precursor of heme. The apparent rate-limiting step in the synthesis of PPIX
is the availability of ALA.148 Therefore, by providing large quantities of exogenous ALA, certain cells will
produce sufficient amounts of PPIX to allow for a PDT effect when illuminated with an activating quantity
of light. Different types of cells appear to have different capabilities for synthesizing PPIX from exoge-
nously provided ALA.148,156 Topical application of ALA to superficial basal cell carcinomas and squamous
cell carcinomas induced PPIX fluorescence and photosensitization restricted to the area of ALA applica-
tion. Kennedy et al. have reported treating more than 300 superficial basal cell carcinomas in patients
using topically applied ALA and broad spectrum red light with a complete response rate at 3 months of
approximately 79%.148 No cutaneous photosensitivity was reported in sites other than those at which
ALA application was given. Recent clinical trials using topically applied ALA appear to corroborate the
initial clinical reports.149,150,156
Wolf et al.149 reported a phase II study in which patients with actinic keratoses, early invasive squamous
cell carcinomas, superficial basal cell carcinomas, noduloulcerative basal cell carcinomas and cutaneous
metastases of malignant melanoma were treated with ALA PDT. ALA was topically applied as a 20% oil-
in-water emulsion for 4 to 8 hours. Approximately 4 hours after application of ALA, photoactivating
visible light was provided by a slide projector with or without a filter, which eliminated wavelengths
below 570 nm. Total applied fluence varied from 30 to 100 J/cm2. CRs were observed in 9 of 9 actinic
keratoses, 5 of 6 (83%) early invasive squamous cell carcinomas, 36 of 37 (97.3%) superficial basal cell
carcinomas, 1 of 10 (10%) noduloulcerative basal cell carcinomas, and 0 of 8 metastatic malignant
melanoma lesions. With a median follow-up of 7 months (range 3 to 12 months), only one recurrence
among complete responders (in a patient with superficial basal cell carcinoma) was seen.
Shanler et al.150 reported that topical application of 20–40% ALA using occlusive dressings in humans
for 4–5 hours resulted in PPIX concentrations (as measured by fluorescence) that were 3–6-fold higher
in actinic keratoses and basal cell carcinomas than in normal skin. Treatment with laser light at 630 nm
to a total fluence of 150–200 J/cm2 was found to be clinically effective for the complete resolution of
superficial lesions. Topical LEVULAN in solution, together with blue light, was approved for marketing
in the United States in 1999 for the treatment of non-hyperkeratotic actinic keratoses of the face and scalp.
10.12.2.1 LEVULAN PDT for Treatment of Actinic Keratoses of the Face and Scalp
LEVULAN Topical Solution, 20%, plus blue light at 6–10.9 J/cm2, has been used to treat actinic keratoses
in 232 patients in six clinical trials.151 Phase III studies were two identically designed, multicenter two-
arm studies using LEVULAN KERASTICK® for Topical Solution applicators plus blue light for 1000
seconds (16 min, 40 sec) for a nominal exposure of 10 J/cm2. Excluded from these studies were patients
who had a history of cutaneous photosensitization, porphyria, hypersensitivity to porphyrins, photoder-
matosis, or inherited or acquired coagulation defects. A minimum of four and a maximum of 15 clinically
typical, discrete, non-hyperkeratotic, target actinic keratosis lesions were identified. Target lesions on the
face or on the scalp, but not in both locations in the same patient, received treatment. The patients were
randomized to receive treatment either with LEVULAN 20% topical solution plus blue light or Vehicle
plus blue light. Patients were randomized at a 3 to 1 LEVULAN to Vehicle ratio. Altogether, 243 patients
were enrolled in the two identical phase III studies and pooled efficacy results at the end of 12 weeks
(lesions remaining at the week 8 follow-up period could be retreated) indicated that 88% of LEVULAN
PDT-treated patients responded as compared with 20% of Vehicle-treated patients (P < 0.001). Patients
did not receive follow-up past 12 weeks after the initial treatment.
No non-cutaneous adverse events were found to be consistently associated with topical LEVULAN
application followed by blue light exposure. The constellation of transient local symptoms of stinging or
burning, itching, erythema and edema as a result of LEVULAN KERASTICK for topical solution plus
blue light treatment was observed in all controlled clinical studies of LEVULAN photodynamic therapy
for actinic keratoses treatment. Stinging or burning subsided between 1 minute and 24 hours after the
BLU-U Photodynamic Therapy Illuminator was turned off, and appeared qualitatively similar to that
perceived by patients with erythropoietic protoporphyria upon exposure to sunlight. There was no clear
drug-dose- or light-dose-dependent change in the incidence or severity of burning and stinging. The
most common changes in lesion appearance after LEVULAN PDT were erythema and edema. In 99%
of active treatment patients, some or all lesions were erythematous shortly after treatment, while in 79%
of Vehicle treatment patients, some or all lesions were erythematous. In 35% of active treatment patients,
some or all lesions were edematous, while no Vehicle-treated patients had edematous lesions. Both
erythema and edema resolved to baseline or improved by 4 weeks after therapy. LEVULAN Topical
Solution applications to photodamaged perilesional skin resulted in photosensitization of photodamaged
skin and a photodynamic response.
10.12.2.2 ALA PDT for Lesions of the Gastrointestinal Tract

Systemic administration of ALA appears to result in the selective synthesis of PPIX within certain mucosal
surfaces. The effects of intravenously administered ALA at various doses on the accumulation of PPIX
in rat colon152 and stomach were studied.153 Fluorescence levels in normal colonic mucosa peaked at 4-
5 hours after administration of ALA. The ratio of fluorescence in the mucosa to that seen in muscle or
submucosal regions was 6:1 at all times,152 and a similar selective PPIX synthesis by rat gastric mucosa
was also observed.153 Selective killing of the rat colon tumor model was seen in this study152 using ALA
PDT, with only normal mucosa showing damage from PDT, while muscle and submucosal tissues
remained intact.152 The remarkable selectivity demonstrated by this system suggests the possibility of
future ALA PDT treatment for mucosal carcinomas with an improved therapeutic index as compared
with PHOTOFRIN. Systemic dosing of patients with ALA has been reported.156 Three patients with
histologically proven carcinomas of the oral cavity were given ALA orally as a bolus dose (30–80 mg/kg).
Peak fluorescence of PPIX was measured in tumor biopsy specimens and was found to peak between 4
and 6 hours after ALA administration, and returned to background autofluorescence levels in 24 hours.
One patient had 60 mg/kg ALA readministered by the oral route followed in 5 h by 630 nm laser light
at 100 J/cm2, which resulted in selective tumor necrosis.
A number of clinical studies have been reported for ALA PDT in Barrett’s esophagus (BE), with only
the largest series reported here. Gossner et al.154 treated 32 patients with ALA-PDT using 60 mg/kg of
ALA given orally and 635nm laser light delivered 4-6 hours after drug dosing through a 2 cm cylinder
diffuser-tipped fiber optic at a power density of 100 mW/cm2 to a light dose of 150 J/cm2. Ten patients
had high-grade dysplasia (HGD), and the remainder mucosal cancer. Patients were maintained on
omeprazole for the duration of the study. Dysplasia was eradicated in all patients with HGD, and there
was complete remission of the cancers in 17 of 22 patients (77%). Of note was that all the tumors that
were not eradicated were greater than 2mm in depth. This remission was maintained during follow-up
of 1–30 months (mean 9.9 months). Re-epithelialization of the Barrett’s mucosa by histologically normal
squamous epithelium was seen in 68%. However, the presence of non-dysplasic specialized columnar
epithelium under the neo-squamous epithelium was noted in some patients. A mean of 1.7 treatment
sessions were required for the eradication of mucosal tumors. Side effects included transient nausea up
to 6 hours after PDT, managed by symptomatic therapy. Mild transient increases in hepatic aminotrans-
ferases (ALT and AST) were noted that returned to normal levels within 1 week and remained normal
throughout follow-up. No patients reported dysphagia or exhibited strictures or systemic photosensitivity.
Ackroyd et al. used green (514nm) light for the treatment of Barrett’s esophagus with LGD.155,157,158
This group also used a lower dose of ALA (30mg/kg) than most other studies. In the only double-blind,
placebo-controlled study reported for BE, 36 patients with low-grade dysplastic BE on chronic omepra-
zole therapy were randomized to receive oral ALA (30 mg/kg) or placebo, followed 4 hours later by
endoscopy and treatment of up to 6 cm of BE with green laser light (514 nm) to a total light dose of 60
J/cm2 through a Perspex cylinder.155 Follow-up endoscopy and biopsy was performed at 1, 6, 12, and 24
months. A response was seen in 16 of 18 patients (89%) in the ALA group, with a median area decrease
of BE of 30% (range = 0-60%). In the placebo group, a median area decrease of 0% was seen (range
0–10%). In the ALA PDT group, the 16 patients maintained regression to normal squamous epithelium
over the entire 24-month follow-up period, and no dysplasia was observed in the treated areas of BE
that remained in any patient. In the placebo group, persistent low-grade dysplasia was found in 12 of 18
patients (67%) followed for 24 months (P < 0.001). The only consistent side effect of ALA PDT seen in
all treated patients was some degree of pain in the chest during light treatment, but analgesia was required
in only 3 of the 18 (17%). The discomfort persisted for 3–5 days following treatment and was aggravated
by swallowing or coughing. One patient developed a mild rash after exposure to sunlight on the day after
treatment. None of the patients developed strictures or complained of dysphagia during the course of
the study. No laboratory abnormalities were reported. Improved methods of circumferential light delivery
may be required for complete ablation of BE.
In the normal rat colon, the area of mucosal necrosis caused by ALA PDT could be increased by a
factor of 3 by interrupting the light dose for 150 sec, after 20% of the total light energy had been
delivered.159 It was hypothesized that fractionation of the light regime for even a short time could
result in replenishing of molecular oxygen, allowing for production of additional singlet oxygen,
whereas continuous illumination caused rapid depletion of oxygen, limiting the PDT effect. In a recent
paper,160 the partial pressure of oxygen (pO2) was measured using microelectrode technology in rat
colon mucosa before, during, and after ALA PDT 1 mm and 3 mm away from the site of irradiation
with a fiber optic. Although a fall in pO2 was observed adjacent to the fiber during continuous and
fractionated light delivery, pO2 fell at the more distant site only during the fractionation regime.
Therefore, light fractionation might increase the efficacy of BE mucosal ablation if the model is
consistent with human photophysiology.
10.13 Conclusions
PDT is now an approved therapy in several areas of medicine. However, PDT continues to evolve and
to have mechanisms of treatment refined and extended. This is most clearly seen in the sections on
intraoperative PDT (abdominal, intrathoracic, and intracranial indications). PDT may have a potential
role in treating peritoneal carcinomatosis and pleural malignancy, but it is in these indications, which
involve treatment of large surface areas during operative procedures, that the need for high output lasers
and improved methods of light delivery are evident. Still, development of new photosensitizers with
increased singlet oxygen production efficiencies and higher wavelengths of activation than 630 nm
continues, which could allow the effective use of existing lasers in even these challenging indications.
The role of PDT in the treatment of head and neck cancers appears to be focused realistically on
treatment of superficial recurrent tumors of the oral mucosa and oropharynx as well as the treatment of
“condemned mucosa.”
Treatment of intracranial tumors remains an exciting area of investigation for PDT, and recent results
suggest a tantalizing connection between light dose and increased survival in non-glioblastoma brain
tumors. However, this area of research requires much work, particularly in the standardization of light
delivery systems and in the development of randomized multicenter clinical trials with stratification
based on tumor type and location.
The number of potential second-generation photosensitizers in clinical trials is increasing, and certain
clinical advantages of some compounds may prove sufficiently attractive to replace PHOTOFRIN in the
future. However, it is likely that different photosensitizers will find optimal uses in certain indications.
For use in intraoperative PDT indications, which require several hours of exposure to laser light (using
present-day technology) it is likely that the ability to be retained in tissues for many hours during which
photosensitizing ability remains maximal (as with PHOTOFRIN) will prove useful. In contrast, for
ambulatory patients with skin lesions, a rapidly clearing topically applied photosensitizer such as LEVU-
LAN is clinically desirable. The next few years may be those in which PDT achieves more of its potential,
with advances in hardware (diode lasers, for example, or the development of useful non-laser light
sources) putting PDT within reach of many more clinical and basic researchers.
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1146-index Page 325 Thursday, November 8, 2001 4:10 PM
Index
A anaerobic, 183
anastomoses, 269, 274, 277
3-amino-1,2,4-triozole, 182 arterio-arterial, 277
abdominal space, 291 anesthesia, 163
ablation, 109, 112 – 114, 116, 120, 123 –127, 138, 164, aneurysms, 276-277
171, 231, angina, 269
234, 236, 256-260, 266, 278 class, 270
arrhythmogenic foci, 271 angiogenesis,
plume, 122, 260 stimulated, 270
threshold fluence, 110-111, 118-119, 122, 125, 128 angioplasty, 155, 262,
absorbance, 90, 138 balloon, 262-265, 267
absorption, 172, 231, 252 coronary(also PTCA), 262
cellular, 92 laser, 248, 250, 252, 255, 260, 263, 264-265, 267, 271
coefficient, 111-117, 120,122, 135, 217, 233 transluminal, 152, 262-265
cross section, 117 percutaneous, 153, 157
deoxyhemoglobin, 162 percutaneous transluminal (PTA), 262, 264-265
depth, 217 peripheral, 262
Dopa-Melanin, 94 anisotropic factor (g), 35
mammalian cells, 94 aorta, 113, 250, 268
molecular, 91, 112 aortotomy, 276
multiphoton, 50, 113, 257 arabinose, 191
oxyhemoglobin, 94, 162 aromatic ring, 112
spectra bands, 179, 182, 215 arrhythmias, 269
tissue, 95, 137, 275 arterial spasm, 153
two photon absorption, 51, 112 arterioles,
urocanic acid, 94, 102 retinal, 213
water, 137 arteriotomy, 276
absorptivity, 112, 253 artery, 258, 260
acetylcholine, 190 brachial, 249, 251
acidification, 184 coronary, 151, 155, 250, 262
acidosis, 189, 201 corotid, 275
acridine orange, 193 femoral, 249, 251
actinic keratoses, 315 peripheral, 262
action, pulmonary, 250, 272
spectra bands, 179 retinal, 239
spectrum, 175, 198 astigmatism, 227, 256
spectroscopy, 96, 102 astrocytoma, 309
acousto-optical, 125 atherosclerosis, 151–150, 262
acuity, 239 atherosclerotic,
adenosine triphosphate (ATP), 172-174, 182, 184, 186- plaque, 248, 252
187, 192, 199 atrium,
adinosine dinucleotide (ADP), 173, 182 left, 249
adrenaline, 190 right, 249
aerobic, 183 attenuation coefficient, 28, 137
albedo, 29, 31-36, 62-63 autocrine, 192
ALA, (d-amino levulinic acid) 145-147, 149, 159, 314-
317 B
amino acids, 112-113, 115 backscatter factor, 62
amplifier, bacteria, 92, 97
Raman, 162 bacteriorhodopsin, 172
325
balloon dilation, 262, 271 pharynx, 302

basal cell cancer, 142,145, 159 skin, 302
basiloma, 147 superficial bladder, 294
basophils, 196 cancer detection
Barrett’s esophagus, 305, 316 endoscopic, 133, 300
Beer’s Law, 28, 36, 42, 90, 113, 116, 118, 139 capsulotomy,
bending posterior, 232
bend radius, 18 carbohydrates, 113
benzene, 112 carcinogenesis, 102-103, 229
bladder, 145, 290 carcinoma,
bladder cancer, 141-142, 145, 291 basal cell, 103, 146, 159, 238, 311, 313, 315
superficial, 294 breast, 144, 313
blackbody, 51 conjunctival, 238
blood, 122, 155, 157, 158, 196 ductal breast, 162
clots, 151 in situ, 142, 291, 294,
vessels, 220, 239, 248, 254 mucosal, 316
Boltzman refractory, 296
constant, 73 squamous, 103, 146, 157, 302, 311, 315
distribution, 1 cardiologist,
bonds, pediatric, 251
peptide, 112 cardiomyocytes,
bone, 161 guinea pig, 193
bowel wall, 306 cardiomyopathy, 272
Bowen’s disease, 311 cardiovascular,
Bowman’s layer, 215, 233 applications, 247
brachytherapy, 229. 310 catalytic activity, 188
brain, 213 cataract, 211, 220, 239, 310
bronchoscopy, 145, 163 formation, 230
bronchus, 289 surgery, 228
breast carcinoma, 144 cataractogenesis, 229
bubble formation, 124 catheter, 248, 260, 262
balloon, 249
fiberoptic, 248
C intravesical laser, 294
Ca, 153 laser delivery, 249
Ca+ , 153 cell
intracellular, 186 death, 100
Ca2+, 182, 193 endothelium, 151, 193
cadmiun chloride, 186 epithelial, 232
calcium, 151, 260 human HeLa, 93
calcium phosphate, 260-261 membrane effects, 101
calcium salts, 266 monkey kidney, 93
cancer, mutation, 100
bladder, 291 CCD, 149
cervical 300 near IR, 157
colorectal, 305-306 chamber,
endobronchial lung, 296, 300 anterior, 213
endometrial, 300 posterior, 213
esophageal, 289, 304 ventricular, 250-251
gastrointestinal, 304 chemiluminescence,
gynecological, 300- 301 luminol-amplified, 182, 193, 196
head, 302, 317 chemotherapy, 294, 305
larynx, 302 chopper
lip, 302 acousto-optic, 50
neck, 302, 317 beam, 54
non-small-cell lung, 296, 300 charge-coupled device, 57
oral cavity, 302 mechanical, 50
ovarian, 300 wheel, 144
Index 327
cholangiocarcinoma, 306 D
cholesterol, 151, 157
chlorin, 139 decay,
chlorophyll, 96, 171-172 constant, 163
chromatin, 193 time, 156
chromophores, 95, 101-102, 112, 115, 117, 118, 120, density,
152, 162, 172, 178, 214, 289 energy, 217, 224, 252
cardiovascular, 248 power, 217, 252, 255, 257
endogenous, 145 dentists, 196
chromatography, 123 detection
choroid, 213, 310 endoscopic cancer, 135
choroidal neovascularization, 314 modulation depth, 160
C. albicans, 102 phase shift, 160
cAMP, 178, 186 detectors
C2, 123 indium gallium arsenide, 52
ciliary body, 213, 224 intensified diode array, 143
circadian rhythms, 198 intensified matrix, 149
clonogenic activity, 181 light, 51
CN, 123 photodiode, 51
CO2, 123 photomultipliers, 52, 53, 144
CO, 123 photothermal, 51
coagulation, 171, 231 silicon, 52
coherence, dermatologists, 196, 256
temporal, 239 dermatology, 137
collagen, 115,120,139, 151, 154,154, 192, 227, dermis, 95
267, 275, 277 Descemet’s membrane, 215
fibrils, 212, 226 delayed contact hypersensitivity, 102
lenticules, 227 delivery systems, 230, 236, 264, 287, 289
submucosal, 306 denaturation, 121
concentration dichroic
molar, 113 beam splitter, 144
conservation of mass, 127 mirror. 141
contractile function, 190 diffuse reflectance, 67
compressive stress, 127 diffusion
colon, 305 approximation, 34-35, 40, 42-43
tumor, 145 constant, 40
cones, 213 diaphanography, 161
conization, 300 di-hematoporphyrins ether (DHE), 238-239, 287
coronary artery, diffraction grating spectrograph, 57
left anterior descending, 269 diopters, 213, 226
cornea, 115,120, 123, 125-126, 211-213, 215, 222, 226, disection, 153
230, 233, 237-238 dispersion, 163
corneal sculpting, 234, 256 dissociation, 118
cough, 300 DNA, 87, 92-93, 96-100, 113, 173, 176, 183-184, 187
cryotherapy, 229, 300, 311 Doppler
cutting, 171 effect, 140, 158
CuA (dinuclear copper), 174, 176, 181 laser, 140
CuB (dinuclear copper), 175-176, 179-181 perfusion monitoring, 136
cyclophotocoagulation, 224, 231 dosimetry
cysteins, 175 light, 294
cytochrome c oxidase, 174-176, 180-182, 184, optical, 47
198-200 probe, 294
cytokines, drug, 287
interleukin-1, 294 safety, 288
tumor necrosis factor, 294 dye, 276
cytoplasm, 98, 184, 186 (also see laser, dye)
cytosol, 174 indocyanine green, 276
cytotoxic action, 289 dysphagia, 289, 304-305
dysplasia, 145, 192 focal volume, 15

dyspnea, 296, 300 fiber,
dysuria, 291 bundles, 19
chalcogenide, 23
cleaved, 304, 306, 308
E connectors, 19
edema, 301, 314, 316 contact tips, 19
elbow, 249 cylindrical-diffuser, 289-290
elastin, 139, 151, 154, 156 cylindrical microlens-tipped, 300, 304
emmetropic eye, 213 damage, 19
endarterectomy, 271, 273-274 fluoride glass, 22
aortic, 273 infrared tramsmitting, 22
iliac, 273 launching energy into, 19
poplitea-posterior tibial, 273 metal-tipped, 255
profunda femoral, 273 microlens, 304
thrombo, 273 optics, 16, 232, 257
superficial fundus, 273 polychrystalline, 23
endothelium, 215, 227 silver halide, 23
cell, 149, 155 single crystal sapphire, 23
endoscope, 143, 291, 316 spherical diffuser-tipped, 291, 294
energy, tips, 289
density, 217 fibrils,
threshold, 114, 125 collagen, 212
enucleation, 229 fibroblasts, 173, 178, 184, 192-193, 201
enzyme, human skin, 185
activation, 181 fibrinogen,
antioxidant, 182 human, 276
cardiac, 269 fistula, 222
fast, 175 flavins, 183
inhibition, 181 flavoproteins, 183-184
native, 195 fluence, 48, 113
slow, 175 rate, 27, 50, 61
eosinophils, 196 threshold, 114
epidermis, 95 fluorescence, 138, 141, 149, 153, 157, 229, 288,
epithelium, 215, 227, 233, 235 306, 315-316
iris pigment, 214 auto, 139, 142-145, 153, 157
retinal pigment, 214, 219 endogenous, 145
erythema, 301, 314, 316 imaging, 149
erythrocytes, 186 probe-beam laser induced, 124,136, 139-141, 153
Escherichia coli, 181, 192-193, 195, 200 fluorescent
esophagus, 289 probes, 60
Barrett’s, 305 Fluorine, 235
eukaryotic cells, 174, 185, 192 fluorosenser, 143
extinction fluoroscopy, 249
coefficient, molar 112-113, 117 free radical,
extracellular pH, 190 formation, 257
reactions, 196
F
G
FDA, 256, 264-265, 278
femtosecond lasers, 25 gastroesophageal reflux (GERD), 305
Ferrocytochrome c, 174 gastrointestinal lesion, 316
fiber lasers, 25 Gaussian,
field, beam profile, 49
visual, 212 pulses, 49, 50
filters, 53 gating
interference, 144 frequency doubling, 162
f-number, 15 glaucoma, 211, 221-222, 224, 240, 310
Index 329
open-angle, 222 aqueous, 211, 213, 221-222, 224

glioblastoma, 307, 309 vitreous, 211, 213, 311
glioma, 308-309 hydrogen peroxide (H2O2), 186
glucose deprivation, 186 hyperopia, 234
glue, 276 hyperopic, 214
glutathione (GSH), 99-100, 185 hyperplasia, 266
glutinin, 193 hyperthermia, 229
glycerol, 190 interstitial laser, 69, 73
glycolysis, 190 hypertrophy, 250
graft, hypoxia, 189, 201
saphenous vein, 277
groin, 249
growth rate, 193
I
Gruneisen stress, 122 illuminating, 231
GSSG, 185 intima, 149
guide wire, 249, 267 intensifier
Gulstrand’s, image, 144
schematic eye, 213 interference fringes, 239
intrathoracic space, 291
imaging
H
laser flash, 123
hand, 161 multi-spectral, 136
Hanks’ solution, 178 radiological, 69-70
haptic loops, 232 time-gated, 162
heart failure, incubation, 128
congestive, 269 indium-antimonide, 70
heat diffusion, 1, 10 indocyanine green, 39
heating, 164 infrared (IR), 136-137, 157, 172, 193, 200-201, 214
HeLa cells, 173, 181, 183-184, 186, 188, 199 ink, 163
Helix pomatia, 186, 193 inorganic salts, 264
hemangioma, 138 imaging, 215, 231
hematocrit fluorescence, 149
hematoma, 277 multi-spectral, 136
heme imidazole, 175
heme a, 174-175, 183 immune effects, 101-103
heme a3, 174-175, 183 immunotherapy, 294
oxygenase, 186 impulse
hemoglobin, 136-139, 145, 157-160, 163, 214, 221, 289 coupling coefficient, 125-126
oxygenated, 139 specific, 125, 127
hemoptysis, 296 inflammation,
HPD (hematophorphyrin derivative), 138-142, 144- chronic, 189
145, 157, 287-288, 296, 300, 302, 304-305, intracellular pH, 189-190
309-310 intraocular,
Herpes simplex, 102 lens, 213
heterodyne, 102, 140, 163 pressure, 213, 222
histidine, 175 structures, 215
HIV, 102 integrating spheres, 53
HO (hydroxyl), 182 intima, 151
H2O, 123 Intralipid, 163, 307
H2O2, 175, 182 Iodine, 263
HCN, 123 iodoacetamine, 186
holography ion exchange, 190
light-in-flight, 163 ionization, 257
homeostasis, ionizing radiation, 157, 164, 229
calcium, 190 iridecrtomy, 231-232
cellular, 184, 186, 200 iris, 213, 221
hot-tips, 262-264 iron-sulfur center, 174, 183
humor, irradiance, 28, 30,32
continuous, 28 semiconductor, 198, 231

pulsed, 28 TmHoCr:YAG, 222, 226, 269, 271
irradiation, titanium, 9
dichromatic, 176, 198 uv ablation, 109
ischemia, 190. 201, 220, 270 vaporization, 311
isobestic point (805nm), 162 xenon chloride (XeCl), 109, 111, 121-122, 124-125, 229,
273
lamp,
K slit, 233
keratinocytes, 192-193, 201 xenon arc, 220
keratoplasty, 226 laparatomy, 307
La Place’s law, 277
Larynx, 302
L lens, 211, 230
lamella, capsule, 228
internal elastic, 273 cataractous, 228-229
Laser interocular, 213, 232
Alexandrite (Cr:BeAl2O4), 9 posterior capsule, 212
argon fluoride (ArF), 109, 111-113, 115-116, 123- lesion
125, 128, 233-234, 254, 256 atherosclerotic, 153
argon-ion, 137, 161, 211, 220-221, 224, 230-231, fibrotic, 154-155
238, 248, 255, 262, 271, 277, 290, 300, 309, LEVULAN, 288, 314-317
314 ligand, 175
carbon dioxide, 6, 8, 224-225, 227, 248, 262, 268, 270- light scattering, 134
271, 273, 277 light source, 287
cavity-dumped, 159 limbus, 222
copper vapor, 290-291 lip, 302
continuous wave (cw) lipids, 112-113, 151
damage, 19 lipofusion, 215
diode, 158, 161-163, 186, 195-196, 211, 224, 230- liver, 288
231, 276, 317 lock-in amplifier (LIA), 54, 56
dye. 137, 211, 220-221, 224, 230, 239, 290-291, 300, lung,
314 cancer, 291
endo systems, 229 tumor, 145
erbium YAG, 6, 217, 225, 232, 254 lymph nodes, 300, 304-305
erbium YSGG, 217, 271 lymphocytes, 193-194
excimer, 211, 235, 254, 257, 264-265, 270, 290-291 lymphoma,
free electron, 14 murine cells, 101
femtosecond, 181 T-cells, 149
gold vapor, 290, 307, 309
HF (hydrogen fluoride), 225 M
holmium, 6, 226, 254, 269
helium-neon, 158, 176, 181-184, 186, 188, 190, 192- macular degeneration, 221, 314
194, 198-199, 233, 239, 289 age-related, 314
infrared, 6 hemorrhagic, 314
krypton, 220-221, 224, 230-231 serous, 314
krypton fluoride (KrF), 115-116, 123 macular
mid-infrared, 211, 224-225, 229 edema, 220, 231
mode-locked, 156 region, 215, 220
near-ir (NIR), 158, 224 macrophages,
neodymium, 1, 157, 211, 224, 230-233, 235, 237, mouse peritoneal, 195
248, 255, 262, 267, 273, 275, 291, 296, 300, magnesium, 174
304, 306 malignancies,
nitrogen, 143, 149, 156 gynecologic, 300
picosecond, 161 malignant, 238, 288
power, 15 tumor diagnostics, 136, 145
probe, 222 mamalian cells, 92. 96, 189, 192
ruby, 1, 219 mammography
Index 331
optical, 135 processes, 110

x-ray, 162 murine spleen cells, 193
materials muscle,
organic, 113 cardiac, 251
melanin, 136, 212, 214-215, 289 musculoskeletal,
melanoma, 103, 238 aches, 196
malignant, 310 pains, 196
ocular, 310 mutation, 97, 100-101
membrane mutagenesis, 98, 99, 160
cellular, 187 myocardium,
mitochondrial, 289 contractility, 269
plasma, 186, 289 ischemic, 268-269
retinal, 232 perfusion, 269
subretinal neovascular, 221 myelocytes, 196
menadione, 186 myoglobin, 272
mercury-cadmium telluride, 70 myopia, 256
mercury lamp, 144 myopic, 213, 235
mesothelioma, 307 myomectomy, 250, 271
metabolism, 185 myotomy, 250, 271
metabolic pathways, 172
metal-ligand systems, 173
metaplasia,
N
intestinal, 305 nanosecond, 233
metastasis, NAD, 139, 142, 185
bladder cancer, 294 NAD+/NADH ratio, 190
breast carcinoma, 144 NADH, 139, 142, 173, 182, 184-185, 200
methionine, 175 NADP, 185, 188
Methylene blue, 188, 190 NADPH, 185, 188
Microwaves, 229 National Cancer Institute (NCI), 307, 313
mid-infrared, 215 National Institutes of Health (NIH), 307
miniaturization, 231 necrosis, 270, 301
minimally invasive, 69-70 ischemic, 288
mirror, nerve
dichroic, 143 Bundle, 239
mitochondria, 172, 182, 184-185, 187, 199, 201 optic, 213
matrix, 174 regeneration, 197
membrane potential, 173, 184 neurons,
optical properties, 173 depolarization, 186
mitogenic signals, 185 rat hippocampus pyramidal, 194
modulation transfer function (MTF), 239 rat spinal cord, 194
Moh’s micrographic surgery, 311 neutrophils, 196
monochromatic, 172 nevus syndrome, 311, 313
monomers, 118 nitric oxide (NO), 195
monochromator, 53 nitrogen laser, 143, 149, 156
monocytes, 196 nucleic acid, 112, 136
mucosa, 291 nucleus, 186
Barrett’s, 316-317 numerical aperture, 16, 58
cervical, 302
colonic, 316
deep gastric, 306
O
necrosis, 317 O2 , 172
vaginal, 302 occlusion, 262, 265
multichannel analyzer ocular,
optical, 143 anatomy, 212
multiphonon physiology, 212
absorption, 50 opacification,
edge, 50 lens capsule, 211, 240
excitation, 255 ophthalmology, 137, 211, 256
ophthalmoscope, 230, 240 etching, 233

scanning laser (SLO), 239-240 excitation, 181, 198
optic nerve, 213 heating, 217
optical multipliers, 144
biopsy, 136 pigments, 215
breakdown, 231 radiation, 308
density, 90 refractive kerotectomy (PRK), 234
mammography, 133 receptor, 172, 201, 213
multichannel analyzer, 57 sensitivity, 195, 287, 304, 310
optically thick, 21 sensitizers, 162, 238, 287, 291, 294, 306-307, 311,
optimal dose, 294 313-315
organic, 117-118 stimulation, 192
organelles, 92 synthetic reaction, 181
ostial lesions, 264-265 thermal, 212, 217, 221, 224, 231
oxidation, 257 toxicity, 238
oxygen, 175, 181, 288-289 vaporization, 217
molecular, 287 welding, 227
singlet state, 138, 183, 288-289 Photofrin, 138, 142, 144, 144-145, 149, 157, 287-290,
tension, 189 294, 296, 300, 302, 304, 307, 309, 311, 313-
oxygenation, 157, 163 314, 317
photon
counting, 158-159, 161
P density, 119
pacemaker, phtallocyanide, 238
electrode leads, 265 phtalocyanines, 139
pain attenuation, 197 physiotherapists, 196
palliation, phytochrome, 172
malignant dysphagia, 304 picosecond, 233
peptide, piezoelectric, 125, 256
bonds, 92, 112-113, 115, 120 pigments, 95,100
carbonyl, 175 Plank’s Radiation law, 70, 151-150, 152,156
perforation, plaque. 135, 149-150, 152, 157, 260-262, 267, 271, 273
vessel wall, 153, 262-263 plasmacytes, 196
perfusion, 158 polymethylmethacralate (PMMA), 115, 124, 128
peristalsis, 306 porphyria, 315
PHA, 194 porphyrin, 173, 183
phacoemulsifiers, 229 port-wine stains, 135
pharynx, 302 polyimide, 116, 120-125,128
photo power density, 48
ablation, 233, 235-236 pressure, 257, 260
absorbing, 171 interocular, 212-213, 222
acceptor, 171, 172,184, 198, 200 piezoelectric sensor, 256
acoustic, 121-122, 125 prokaryotic cells, 192
activating, 146, 238 prosratic gland, 143
activating, retinal, 71 proteins, 92-93, 100,112-113, 120, 136, 173, 183,
activating, enzyme, 103 275-276
bleaching, 289, 310 stress, 186
chemistry, 164, 171, 199, 201, 212, 254, 274 synthesis, 195
chemotherapy, 287 proton motive force, 184
coagulation, 69, 215, 217, 219-221, 224, 229-232, protoporphyrin, 145, 314
240, 310-311 psoralens, 171
cutting, 224 pulse duration, 224
decomposition, 122, 255-257, 261, 264 pulsed photothermal radiometry (PPTR), 68
dematosis, 315 pump sources
disruptive, 212, 215, 221, 231-233, 255 chemical reactions, 5
dynamic therapy (PDT), 69,138, 145, 147, 159, 163, discharge, dc, 4
171, 185, 229, 238-239, 287-291, 294, 296, discharge, rf, 4
300-302, 304, 305, 307, 309-311, 313-317 electron beam, 5
Index 333
electric current, 5 spatial, 148

flashlamp, 3 spectral, 148
laser, 4 restenosis, 264-265
nuclear, 5 reticuloendothelial system, 288
pupil, 213 retina, 213, 215, 219-220, 238-239
PUVA, 171-172 retinal detachment, 310
pyrimidine dimer, 96, 98, 100 retinaoblastoma, 229, 311
pyrometer, 70 retinopathy,
proliferative diabetic, 220
revascularization,
Q transmyocardial laser (TMLR), 268
quantum efficiency, 238 rheumatologists, 196
quantum yield, 90 rheumatism, 190
rhodamine 123, 173
rhodopsin, 171-172, 215
R RNA, 113, 173, 193-194
radiance, 48 rose-bengal, 238
radiation, rRNA, 194
hardening, 128
ionization, 229 S
therapy, 302
radicals, 123 saturation, 180
radiant scattered light, 28
exposure, 28 scattering,
radiation therapy, 311 coefficient, 163
radio frequency, 229 elastic, 139, 157-160
radiologic imaging, Raman, 140
methods, 79 Schwann cells, 178
Raman sclera, 212, 222
amplifier, 162 Seebeck,
spectroscopy, 137, 141, 157 voltage, 74
Rayleigh scatter, 21 coefficient, 74
reciprosity rule, 200 Seldinger technique, 249
rectum, 305 sensors,
redox, integrated circuit, 74
chain, 172 sensitivity, 140, 178
chemistry, 174, 181 sensitization, 231
light-activated, 187 sensitizers, 148
shift, 190 septa,
state, 185-186, 189-190, 192, 200-201 interatrial, 250
reflectance, interventricular, 250
spectroscopy, 137, 157 septostomy, 250-251, 271-272
reflection, 29 atrial, 272
Fresnel, 17, 29-30 septum, 250
Specular, 29, 32 interventricular, 251
total internal, 16 posterior ventricular, 249
reflectometry, shock wave, 125-127, 231, 248, 256-257, 261, 265,
spatial domain, 163 270-271
total internal, 16 shrinkage, 231
refractive index, 113, 212, 215 single photon counting, 60
rehabilitation clinics, 197 sinus,
resonators, 6 coronary, 268
stable, 6-7 coronary retroperfusion, 268
unstable, 6-7 simulations
resonant cavity, 1 Monte Carlo, 29, 31, 34-36, 38, 65
resistive slitlamp, 221, 223, 230
temperature detectors, 74 Soret band, 176
resolution, spasm, 151
spleen, 288 temperature probes

Snell's law, 16, 29, 31 fiber optic, 77
solder, 276-277 thermal
specificity, 140. 157 conductivity, 41, 126
spectrometer, 143 damage, 229, 233, 253, 271
spectrophotometer, diffusivity, 121, 217
double beam, 178 relaxation time, 110, 122, 217
spectroscopy response, 41
fluorescence, 240 thermography, 69
Fourier transform, 156 thermotherapy,
infrared, 123 interstitial, 69
mass, 123 thermisters, 74-76
31P magnetic resonance, 309 thermocouples, 73-74, 76
Raman, 135, 141, 157-157, 240 thoracoscope, 270
FT Raman, 240 thoracotomy, 307
reflectance, 137, 157 thrombectomy, 274
time of flight, 124 thrombosis, 248, 265, 274
time-resolved, 160 thromboxane, 288
specular reflection, 29, 33, 41, 58 tissue, 113
sports medicine, 197 myocardial, 155
Steinhart-Hart equation, 75 optical properties, 289
stain, tomography,
port-wine, 138 computed, 309
stenosis, 277 topical,
aortic, 271 application, 146
subaortic hypertrophic, 272 trabeculoplasty, 221, 231
mitral, 271 trabecular meshwork, 213-214, 221
stents, 266 transcleral, 222
stimulation, transverse mode, 49
lymphocyte, 294 transillumination, 157-158, 160-161
macrophage, 294 transmyocardial, 271
squamous cell carcinoma, 146, 157 transurethral,
strain-to-failure, 18 laser coagulation, 72
stress, trephine, 224
oxidant, 186 triplet state, 138
protein, 186 trypan blue, 178
stroma, 215, 226, 260 tryptophan, 112
corneal, 226, 235 tumor, 149, 159, 161, 306
Stefan-Boltzmann, colon, 145
law, 70 cutaneous, 311
constant, 70 endobronchial, 296
stratum corneum, 95 endotracheal, 296
streak-camera, 162 esophageal, 304
superoxide, 182 intrachranial, 310, 317
dismutase, 188 malignant, 138, 144, 157
surgery, malignant brain, 309
general, 137 malignant intercranial, 307
cataract, 227 margins, 288
corneal, 227 papillary, 291
glaucoma, 227 subcutaneous, 311
ophthalmic, 240 tyrosine, 112
vitreoretinal, 227
sutures, 232, 277
systemic, 146
U
ultrasonic,
probes, 229
T
ultrasound,
tamped conditions, 126 intraluminal, 264
Index 335
ultraviolet, 85-87, 98, 100, 102-105,109, 113, 115, 120, cavity, 268
122,126,138, 152, 172, 229, 234, 254-257, left, 249
260, 270, 274, 306 right, 249, 271
A (UV-A), 85, 88-89, 95-100, 102-104, 171, 186 wall, 268
absorption, 89-90
B (UV-B), 85, 87,89, 92, 97-100, 102-104 vibrational,
C (UV-C),85,87. 89, 92, 94, 96-100, 102-104, 234-235 shifts, 156
direct effects, 96 transitions,157
fluorescent sources, 88-89 viruses, 92
germicidal sources, 88-89 visible, 172, 186, 200-201, 214
halogen lamps, 89 vision, 215
ionizing, 91 visual field, 212
indirect effects, 96 VISUDYNE, 288-289, 314
laser, 88 vitreous, 232
non-ionizing, 91 hemmorage, 231
solar simulators humor, 212-213
synchrotrons, 88-89 vocal cords, 302
sources, 88-89 vulva, 300, 302
terestrial solar, 87-89, 102 cancer of, 300
vaccum (VUV), 85, 87, 89, 94, 96-100, 102-103
umbonate, 94
urethera, 291
W
urinary bladder, 291 waveguides,
uvea, 214 hollow, 24
uveal, wavelength, 252
melanoma, 229 water, 112, 136, 215
tract, 213 Wein Displacement Law, 70
welding, 231, 271, 274-277
dermatology, 227
V
neurology, 227
vagina, 300, 302 otolaryngology, 227
cancer of, 300 urology, 227
valve, vascular, 227
aortic, 249 wound healing, 189, 197
mitral, 250
prostheses, 271
tricuspid, 250
X
valvuloplasty, 271 xanthophyll, 214
valvulotomy, 271 macular, 215
pulmonic, 251 pigment, 220
tricuspid, 251 xeroderma pigmentosum cells, 98, 104
vaporization, 69, 171
veins, 239, 250
pulmonary, 249
Y
vein grafts, 265 yeast, 182-183, 195
velocimetry,
laser Doppler (LDV), 239
vena cavae, 250
Z
inferior, 249, 251 zinc, 174
superior, 249, 251 zonules, 228
ventricle,

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Foreword by the late

Dr. Leon Goldman

Library of Congress Cataloging-in-Publication Data

Lasers in Medicine / edited by Ronald W. Waynant.

Visit the CRC Press Web site at www.crcpress.com

© 2002 by CRC Press LLC

No claim to original U.S. Government works

To my mother, father, wife and children

The Early Development

The Current Laser Theme

Leon Goldman, M.D.

Thomas P. Coohill George H. Pettit Brian C. Wilson

2 Optical and Thermal Response of Tissue to Laser Radiation

3 Dosimetry and Thermal Monitoring

4 Uses and Effects of Ultraviolet Radiation on Cells and Tissues

4.2. A Division of the Ultraviolet for Photobiological Studies ...................................................... 86

5 The Physics of Ultraviolet Laser Ablation

6 Tissue Diagnostics Using Lasers

7 Low-Power Laser Effects

8 Therapeutic and Diagnostic Application of Lasers in Ophthalmology

9 Cardiovascular Applications of Lasers

10 Lasers in Photodynamic Therapy

1.1 Laser Principles....................................................................... 1

1.1 Laser Principles

1.2 Laser Materials

feedback and mirror

FIGURE 1.1 Basic laser components.1

FIGURE 1.2 Population inversion.1

Inverted laser medium

Input amplified output

FIGURE 1.3 Laser Amplifier.1

Inverted laser medium

FIGURE 1.4 Laser oscillator.1

TABLE 1.1 Laser Materials2

1.3 Pump Sources

FIGURE 1.6 DC discharge system.

FIGURE 1.7 RF discharge excitation.

FIGURE 1.8 E-beam system for laser excitation.

TABLE 1.2 Major Chemical Lasers2

1.5 Major Types of Lasers

R1 (Mirror radius) = (a) Plane-parallel R2 =

R2 > (b) Large-radius mirrors >> L

R1 > L (e) Concave-convex R2 = - (R1 - L )

FIGURE 1.9 Examples of

negative branch confocal

positive branch confocal

FIGURE 1.10 Examples of unstable resonators.

FIGURE 1.11 Energy levels of the CO2 molecule.3

FIGURE 1.12 Energy levels of the Nd: YAG laser.3

TABLE 1.3 Neodymium Laser Performance2

Pulsed quad Nd glass Flashlamp 263–266 0.04

FIGURE 1.13 Energy levels of several vibronic lasers.2

TABLE 1.4 Vibronic Lasers

Alexandrite Arc lamp CW 730–810

FIGURE 1.14 Diode laser structures.

TABLE 1.5 Semiconductor Laser Performance

TABLE 1.6 Metal Vapor Lasers

Copper 511, 578 1

Laser Alternative path through

Ground state (S0) Radiationless

FIGURE 1.15 Typical dye laser energy levels.2

Cavity focusing mirror

Dye cell or jet