Part ii Examining and Imaging the Cornea and External Eye
Section 3 Imaging Techniques of the Cornea
Chapter 15
Confocal Microscopy
W. Matthew Petroll, H. Dwight Cavanagh, James V. Jester
Key Concepts
• Confocal microscopy provides high resolution images
of all corneal cell layers in vivo.
• Confocal microscopy through focusing, can be used for
three-dimensional assessment of corneal wound
healing.
• A range of corneal infections can be diagnosed and
tracked temporally using confocal imaging.
• Confocal microscopy is now widely used for measuring
changes in the density and organization of corneal
sub-basal nerves.
• Changes in corneal sub-basal nerve structures
identified with confocal imaging may be an early
indicator of peripheral neuropathy in diabetes.
• Multiphoton-second harmonic generation confocal
imaging is a more advanced technology that allows
noninvasive assessment of corneal collagen matrix
organization in ex vivo tissue.
Background
It is well established that confocal microscopy provides
higher-resolution images with better rejection of out-of-
focus information than conventional light microscopy. The
optical sectioning ability of confocal microscopy allows
images to be obtained from different depths within a thick
tissue specimen, thereby eliminating the need for processing
and sectioning procedures. Thus, confocal microscopy is
uniquely suited to the study of intact tissue in living sub-
jects. In vivo confocal microscopy has been used in a variety
of corneal research applications on experimental animals
since its development over 25 years ago. In recent years, the
use of the confocal microscope on human patients has
also expanded dramatically. In this article we review the
latest developments and most current applications of clini-
cal confocal microscopy of the cornea. In addition, we also
discuss multiphoton-second harmonic generation confocal
imaging, a more advanced technology that allows noninva-
sive assessment of collagen matrix organization in intact
corneal tissue.
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Historical overview
The optical design of confocal microscopy is based on the
principle of Lukosz, which states that resolution may be
improved at the expense of field of view.1 In 1955, Marvin
Minsky developed the first confocal microscope, which was
used for studying neural networks in the living brain.2 In
modern confocal microscopy, a point light source is focused
onto a small volume within the specimen, and a confocal
point detector is used to collect the resulting signal. This
technique results in a reduction of the amount of out-of-
focus signal from above and below the focal plane which
contributes to the detected image, and produces a marked
increase in both lateral (x, y) and axial (z) resolution.3
The use of a point source/detector in the confocal optical
design trades field of view for enhanced resolution; there-
fore, a full field of view must be built up by scanning. Petran
et al.4,5 developed the first scanning confocal microscope,
named the tandem scanning confocal microscope (TSCM),
which used a spinning disk containing thousands of opti-
cally conjugate (source/detector) pinholes arranged in Archi-
medean spirals (Fig. 15.1A). In 1986, Lemp et al.6 were the
first to apply confocal imaging techniques to the study of
the cornea ex vivo. This work led to the design of a TSCM
with a horizontally oriented objective (Tandem Scanning
Corp, Reston, VA), which was the first system suitable for
use in ophthalmology.7
Current confocal systems in clinical use
Three main confocal imaging systems have been used
clinically: the TSCM, the HRT III with Rostock Corneal
Module (HRT-RCM) from Heidelberg Engineering, and the
Confoscan 4 from Nidek, Inc. Much of the in vivo imaging
to date has been accomplished with the TSCM design. Most
TSCM systems currently in use employ a specially designed
surface contact objective (24×, 0.6 NA, 1.5 mm working
distance). The position of the focal plane relative to the
objective tip is varied by moving the lenses within the
objective casing (Fig. 15.1B). Thus, the depth of the focal
plane within the tissue can be calibrated, and quantitative
three-dimensional imaging is possible with this system.8,9
With this objective, the TSCM has an axial (z-axis) resolution
of approximately 9 µm.8 The TSCM system is no longer com-
mercially available.
 CHAPTER 15
Confocal Microscopy

Chapter Outline
Background
In Vivo Confocal Imaging Techniques
Clinical Applications
Advanced Technology: Second Harmonic Generation Imaging
of Corneal Collagen Architecture
Conclusions

180.e1
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1 2 3 4 5 axial resolution than the TSCM, due to the higher NA
A 6
B (Fig. 15.2E).
Fig. 15.1 The tandem scanning confocal microscope (TSCM).
(A) An illustration of the optical pathway used in TSCM. Light from a
broadband source (1) passes through the pinholes on one side of a
Nipkow disk (2) and a beam splitter (3), and is focused by an objective
lens (4) into the specimen (5). The reflected or emitted signal is then
reflected by the beam splitter (3) and front surface mirror (6) to the
conjugate pinholes on the opposite side of the disk, which prevent light
from outside the optical volume from reaching a camera or eyepiece.
Rotation of the disk results in even scanning of the tissue in real time.
(B) A simplified sketch of the TSCM objective, demonstrating how the
depth or z position of the focal plane can be changed by moving the
internal lenses. This allows acquisition of a series of optical sections
through a single cell or other tissue structure. (A from Cavanagh HD,
Petroll WM, Alizadeh H, et al. Clinical and diagnostic use of in vivo
confocal microscopy in patients with corneal disease, Ophthalmology
100:1444–54, 1993. B from Petroll WM, Cavanagh HD, Jester JV.
Three–dimensional reconstruction of corneal cells using in vivo confocal
microscopy, J Microsc 170(3):213–9, 1993.)
The HRT-RCM is a laser scanning confocal microscope. It
operates by scanning a 670-nm laser beam (<1 µm diameter)
in a raster pattern over the field of view. The system typically
uses a 63× objective lens (0.95 NA), and provides images
that are 400 µm × 400 µm in size. The microscope produces
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CHAPTER 15
Confocal Microscopy
images with excellent resolution and contrast, and has better
objective.10,11
The Confoscan 4 is a variable-slit, real-time scanning con-
focal microscope.12,13 The system uses a 40× objective lens
(0.75 NA), and digitized images are 460 µm × 345 µm in size.
This is a user-friendly instrument that incorporates auto-
mated alignment and scanning software. In addition, the
scanning slit design allows better light throughput and pro-
vides images with a higher signal to noise ratio than the
TSCM. However, this is achieved at the expense of axial reso-
lution, which has been measured at approximately 26 µm.14
In Vivo Confocal Imaging Techniques
Normal corneal structures
Normal corneal anatomy as observed with the TSCM is
shown in Figure 15.2.15 Confocal images are always taken en
face: that is, the viewer sees thin slices of the cornea that are
parallel to the epithelial surface. The borders of the surface
epithelial cells are readily seen, as are the bright cell nuclei
(Fig. 15.2A). Immediately beneath the basal epithelium, a
fine nerve plexus can be detected (Fig. 15.2B). In the corneal
stroma, only cell nuclei are visible using TSCM under normal
conditions, with a dark background in between (Fig. 15.2C
and D). Interestingly, the interconnected cell processes of
the keratocytes become visible under certain pathologic con-
ditions, possibly due to tissue edema or cell activation. Large
numbers of keratocytes are present in the anterior stroma as
compared to the mid and deeper stroma, which show a
lower cell density.16 Prominent nerve fibers can be seen
within the stroma, and tracked over long distances three-
dimensionally. TSCM images of the normal endothelium
appear similar to what is observed using specular microscopy
HRT-RCM images of a normal human cornea are shown
in Figure 15.3. Due to its higher signal-to-noise ratio and
improved axial and lateral resolution, intraepithelial section-
ing can be achieved, and wing cells (Fig. 15.3A) and basal
cells (Fig. 15.3B) can be easily distinguished. Langerhans
cells can also be identified and their density quantified using
the HRT-RCM microscope.17 Images of the sub-basal nerve
plexus (Fig. 15.3C), stromal cells (Fig. 15.3D and E) and
corneal endothelium (Fig. 15.3F) are generally similar to that
observed with the TSCM, albeit with higher contrast.
The Confoscan 4 also provides images of all corneal cell
layers, albeit with less resolution than the HRT-RCM. The
Confoscan 4 is particularly well suited to imaging the corneal
endothelial cells. Due to its thicker optical volume, full-field
images are easily obtained. Unlike specular microscopy, the
optical sectioning capability of confocal microscopy allows
imaging through edematous corneas.
Confocal microscopy through-focusing
To collect and quantify 3-D information from the cornea,
a technique termed confocal microscopy through-focusing
(CMTF) is typically used. CMTF scans are obtained by focus-
ing through the cornea from the epithelium to endothelium
181
 PART ii Examining and Imaging the Cornea and External Eye

Section 3 Imaging Techniques of the Cornea

A B C

D E F
90 A
Fig. 15.2 Corneal images digitized directly from a CMTF scan (A–E) and the
corresponding reconstruction (F) and CMTF intensity curve (G) in a human
volunteer. (A) Epithelial image corresponding to peak A; (B) basal–epithelial
nerve plexus image corresponding to peak B; (C) image of anterior layer of
70 C
keratocyte nuclei corresponding to peak C; (D) stromal image corresponding
Image intensity

B to position D; (E) endothelial image corresponding to peak E; (F) 3-D

E reconstruction. (G) CMTF intensity curve. Horizontal field width (A–E) = 330 µm.
(Reproduced from Li HF, Petroll WM, Møller–Pederson T, et al. Epithelial and
D corneal thickness measurements by in vivo confocal microscopy through
50
focusing (CMTF). Curr Eye Res 1997, 16:214–21.)

30
-20 80 180 280 380 480 580
G
Depth (mm)

at a constant lens speed, while continuously digitizing
images. The CMTF intensity curve is then generated by cal-
culating the average pixel intensity in a central region of
each image, and plotting versus z depth (Fig. 15.2G). After
a z series of CMTF images have been digitized, a cursor can
be moved along the intensity curve as corresponding images
are displayed. In this way, the user can identify images of
interest and record their exact z axis depth.18,19 Two major
peaks corresponding to the superficial epithelium anteri-
orly (Fig. 15.2A) and the corneal endothelium posteriorly
(Fig. 15.2E) are present in the normal human cornea, as
well as smaller peaks corresponding to the basal corneal
epithelial nerve plexus (Fig. 15.2B) and the anterior layer of
corneal keratocytes (Fig. 15.2C). By measuring the distance
between the various peaks, accurate and reproducible mea-
surements of corneal, epithelial, and stromal thickness can
be obtained.18 CMTF scans can also be used to generate a 3-D
image of the cornea by stacking the images and projecting
them using surface or volume rendering (Fig. 15.2F).
As mentioned previously, the TSCM uses an applanating
objective, which stabilizes the cornea, and the position of
the focal plane within the tissue is changed by moving the
lenses within the objective casing (Fig. 15.1B). A stack of
evenly spaced CMTF images can thus be obtained and recon-
structed. The HRT III also uses an applanating tip to provide
stability, and cross-sections with excellent resolution and
contrast can be generated. Unfortunately, automated scans
of only 80 µm can be generated using the commercially
available system, and changing the focal plane over larger
distances must be performed manually (by rotating the
objective housing by hand). A modified prototype that
overcomes this limitation and allows CMTF scanning has
recently been developed and applied to the rabbit cornea
(Fig. 15.4A).20 These datasets can be used for interactive

182
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 CHAPTER 15
Confocal Microscopy

A B C

D E F

Fig. 15.3 Images of a normal human cornea obtained using the HRT III. (A) Epithelial wing cells. (B) Basal epithelial cells. (C) Sub-basal nerve plexus.
(D) Anterior stroma just below Bowman’s layer. (E) Mid stroma. Note decreased density of keratocyte nuclei as compared to (D). (F) Normal
endothelium. Horizontal field width = 400 µm.

A 80µm B 80µm C 50µm

Fig. 15.4 (A) 3-D reconstruction of a CMTF scan in a normal rabbit cornea obtained using a prototype HRT–RCM system. (B&C) Volume renderings
of HRT–RCM CMTF data. Images were cropped in 3-D to focus on a region of interest, and rendered using an orthogonal maximum intensity
projection within the Surpass module of Imaris. (B) In this rabbit, there was minimal x−y movement of the cornea during the scanning process
(maximum drift of less than 10 microns), and the reconstruction was made without aligning the images within the stack. (C) In this rabbit, there was
more x−y movement of the cornea during this scan (a maximum drift of 78 µm), thus the planes within the stack were registered using the linear stack
alignment plug–in in image J prior to performing the reconstruction. (Adapted from Petroll WM, Weaver M, Vaidya S et al. Quantitative 3-dimensional
corneal imaging in vivo using a modified HRT–RCM confocal microscope. Cornea 2013;32:e36–43.)

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 PART ii Examining and Imaging the Cornea and External Eye
Section 3 Imaging Techniques of the Cornea
visualization of corneal cell layers, quantitative assessment
of sub-layer thickness and depth-dependent measurements
of keratocyte density. Because of the enhanced contrast as
compared to the TSCM, orthogonal 3-D projections through
the full thickness cornea can also be generated from the
CMTF stacks (Fig. 15.4B and C). Overall, these modifications
have the potential to significantly expand the capabilities of
the HRT-RCM for quantitative research applications.
The Confoscan system uses a noncontact objective lens
(for patient comfort), and movement of the entire objective
lens is used to change the focal plane position and generate
CMTF scans. A trade-off with this design is that the cornea
can move randomly with respect to the lens tip during a
CMTF scan. A “Z-Ring” which touches the corneal surface
can be used to allow accurate calculation of z-axis position
within the cornea during scanning,21 but the distance
between images in CMTF scans is generally not as uniform
as that obtained using applanating objectives. Thus, both
the stability and axial resolution of the Confoscan 4 are
reduced as compared to the TSCM and HRT-RCM.
Clinical Applications
Although confocal microscopy has been used extensively in
research applications on experimental animals, its noninva-
sive nature makes it ideally suited for clinical use in ophthal-
mology.15 In recent years, the clinical application of in
vivo confocal microscopy has expanded rapidly. Confocal
microscopy has been used to monitor changes in keratocyte
density during aging, in keratoconus patients, and following
surgery.16,22–25 The effects of contact lens wear on the mor-
phology and thickness of the corneal epithelium have also
been quantified, and such studies have provided important
insight into how lens type and wear pattern influence bacte-
rial binding and corneal epithelial homeostasis.26–30 There
are numerous other applications of this technology in the
literature which cannot be covered here due to space limita-
tions; many of these are discussed in recent review arti-
cles.26,31–33 Below, we discuss three of the most common
applications of clinical confocal microscopy in more detail:
(1) assessment of wound healing following injury or refrac-
tive surgery, (2) diagnosis of corneal infections, and (3)
changes in the density and organization of sub-basal nerves
in response to surgery or disease.
Wound healing following injury or refractive surgery
Because of its unique ability to image the cornea four-
dimensionally at the cellular level (x, y, z, and t), confocal
microscopy is ideally suited to monitoring the cellular events
of epithelial and stromal wound healing, particularly follow-
ing refractive surgical procedures.34,35 For example, confocal
microscopy allows measurement of wound gape, the depth
of epithelial ingrowth, and the degree of corneal fibrosis in
radial keratotomy wounds.9 Similar assessments can also be
performed following penetrating keratoplasty (PK),36,37 or
lacerating injury. As detailed below, confocal microscopy is
especially well suited to assessing the corneal response to
photorefractive keratectomy (PRK) and laser assisted in situ
keratomeliosis (LASIK). It has also been applied following
related procedures such as LASEK38,39 and epi-LASIK,40 as well
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as other surgical techniques such as Descemet stripping with
automated endothelial keratoplasty (DSAEK).41
PRK: CMTF has been used to assess multiple parameters
associated with corneal wound healing following PRK.42
Changes in corneal, epithelial, and stromal thickness fol-
lowing surgery can be made from the CMTF curves. In addi-
tion, confocal microscopy can be used to assess the degree
of subepithelial haze induced by the procedure. Confocal
microscopy following PRK has demonstrated that the devel-
opment of corneal haze is correlated with the activation
of corneal keratocytes and transformation to a fibroblast
or myofibroblast phenotype.43,44 These activated cells are
more reflective than quiescent corneal keratocytes, and syn-
thesize extracellular matrix components that also reduce
corneal transparency. As shown in Figure 15.5, a “haze peak”
is observed in the CMTF curves after PRK, and the width
and height of the haze peak indicate the thickness and
reflectivity of the subepithelial tissue. An objective haze esti-
mate can be obtained by calculating the area under the haze
peak for each patient. Interestingly, increased subepithelial
haze has been detected using CMTF in patients who were
graded clinically clear using the slit lamp, demonstrating
the higher sensitivity of confocal microscopy.42 Temporal
changes in keratocyte density have also been documented
following PRK.22 Overall, in vivo confocal microscopy
represents an important tool for quantitative assessment of
initial photoablation depth, temporal changes in epithe-
lial, stromal, and corneal thickness, nerve regeneration,45,46
cell loss and/or repopulation, and unbiased haze estimation
after PRK.
LASIK: Confocal microscopy has also been used to assess
numerous parameters following LASIK, such as epithelial
thickness, flap thickness, interface particle density, keratocyte
density, nerve damage and recovery, stromal cell activation,
and interface haze.23,38,46–59 Confocal microscopy has been
used to assess the corneal response to both microkeratome-
assisted LASIK, and LASIK with flap creation using IntraLase
(IntraLASIK). Following traditional LASIK with a micro-
keratome, corneal haze is not generally observed clinically.
Although regions of keratocyte activation and changes in
keratocyte density have been observed by confocal micros-
copy, the overall stromal wound healing response is much
less pronounced than that of PRK.23,47–50
Previous clinical studies have shown that femtosecond
laser enabled flap creation provides fewer complications
than mechanical microkeratomes, and results in better
visual outcomes in most patients. Consistent with these
results, confocal imaging has demonstrated that the femto-
second laser provides more accurate and reproducible flap
thickness, as well as a significant reduction in the number
of interface particles detected.54,60–64 Early confocal microscopy
studies following IntraLase identified keratocyte activation
in some patients, particularly at higher raster energies.62–65
An example of a cornea with keratocyte activation and ECM
haze is shown in Figure 15.6. Note the areas of increased
reflectivity near the flap interface (Fig. 15.6A, arrows). Exami-
nation of individual images revealed keratocyte activation
near the interface, as indicated by highly reflective nuclei
(Fig. 15.6B and C, arrows). An increase in ECM reflectivity
(ECM haze) was also observed surrounding the cells in many
cases. In addition, cell processes were sometimes visible,
 CHAPTER 15
Confocal Microscopy

A B C

haze
200
Image pixel intensity

150 epi
haze

100 epi epi

haze endo
endo
endo
50
A B C

0 100 200 300 400 500 0 100 200 300 400 500 0 100 200 300 400 500

Distance (µm) Distance (µm) Distance (µm)

Fig. 15.5 Three-dimensional reconstructions and corresponding confocal microscopy through–focusing (CMTF) scans of three human corneas one
month after photorefractive keratectomy. (A) A clinically clear cornea (grade 0 haze). (B) Cornea with clinical grade 2 haze. (C) Cornea with clinical grade 4
haze. Note the increased subepithelial reflectivity in all three corneas. In the corresponding CMTF scans, a profound increase in both haze thickness (haze
peak width) and haze intensity (haze peak height) is seen with increasing clinical grades. (From Møller-Pederson T, Vogel M, et al. Quantification of stromal
thinning, epithelial thickness, and corneal haze after photorefractive keratectomy using in vivo confocal microscopy. Ophthalmology 1997, 104:360–8.)

Fig. 15.6 (A) CMTF stack taken 3 months after

LASIK with IntraLase. Note the areas of increased
reflectivity near the flap interface (arrows). (B)
Single image taken from the CMTF stack in A, at
the level of the flap interface. Note highly reflective
keratocyte nuclei (arrows), indicating cell
activation. An increase in ECM reflectivity (ECM
haze) is also observed surrounding the cells (C)
Single image from the CMTF stack taken 30 µm
below the interface. A few activated keratocytes
(arrow) and ECM haze is detected. Horizontal field
width = 375 µm. (From Petroll WM, Goldberg D,
B Lindsey SS, et al. Confocal assessment of the
corneal response to intracorneal lens insertion and
LASIK with flap creation using IntraLase. J
Cataract Refract Surg 32:1119–28, 2006,
copyright Elsevier.)

C
A

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 PART ii Examining and Imaging the Cornea and External Eye
Section 3 Imaging Techniques of the Cornea
suggesting local stromal edema and/or fibroblast transforma-
tion of corneal keratocytes.
Infectious keratitis
Because it provides much higher magnification than a slit
lamp biomicroscope, confocal microscopy is ideally suited
for early detection and diagnosis of a number of infectious
organisms (for reviews, see references 26, 31, 32, 33, and 66).
One important clinical application of the confocal micros-
copy is the localization of Acanthamoeba cysts and tropho-
zoites in the living eye for diagnosis and assessment of the
efficacy of ongoing medical treatment.15,66–72 Figure 15.7A
shows easily identifiable brightly reflective cysts (1) in a
human patient who had a biopsy confirming Acanthamoeba
infection. Figure 15.7B is from a patient who was being
treated with Brolene (propamidine isetionate), and was
showing symptoms of an active infection. Note the appear-
ance of two types of highly reflective structures, presumably
cysts (1) and trophozoites (2). The three-dimensional nature
of this process can be assessed using confocal imaging; thus,
the total spatial volume delineated by the infection can
be quantitated, and response to antiamoebal drug therapy
monitored.
The detection of fungal keratitis has also become an
important clinical application of confocal microscopy, due
in part to the increased incidence of these infections.73 Con-
focal microscopy has been shown to provide distinctive
images of the filaments of Fusarium solani (Fig. 15.8)66,74 and
1
2
1
Fig. 15.7 Acanthamoeba keratitis. (A) Easily identifiable brightly
reflective cysts (1) in a human patient who had a biopsy confirming
Acanthamoeba infection. (B) TSCM image from a patient who was
being treated with Brolene, and was showing symptoms of an active
infection. Note the appearance of two types of highly reflective
structures, presumably cysts (1) and trophozoites (2). Horizontal field
width = 350 µm.
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Aspergillus keratitis66,75,80 in human patients, and may provide
a unique means for early detection of these infections. Elon-
gated particles resembling Candida pseudofilaments have
also been identified in infected corneas.73 Other studies have
demonstrated the potential usefulness of confocal microscopy
in diagnosing bacterial contact lens-related keratitis,76 micro-
sporidial keratitis,77,78 and Borrelia keratitis.79 It should be
noted that not all infectious agents can be detected and/or
distinguished using confocal microscopy, and physical
biopsy of the cornea is still often necessary to confirm diag-
nosis.31,66,70 Further studies are needed to clarify which infec-
tious organisms can be reliably detected and distinguished
using confocal microscopy.
Imaging changes in the sub-basal nerve plexus
An important feature of in vivo confocal microscopy is the
ability to obtain high-resolution images of the sub-basal
nerve plexus, particularly with the HRT-RCM (Fig. 15.3C).
Several studies have used confocal microscopy to assess tem-
poral changes in the density and organization of sub-basal
nerves in response to surgery or disease.38,81–83 In temporal
studies following PRK, regenerating sub-basal nerve fibers
were detected as early as seven days after PRK, but required
at least 1–2 years for complete recovery, perhaps due to the
persistence of the subepithelial scarring.45,46,84 Following
LASIK, sub-basal nerve density remained lower for 3–5 years
after surgery.84
Confocal microscopy of the sub-basal nerve plexus has
been increasingly used as an indicator of peripheral neuropa-
thy in diabetes and other conditions which can affect small
nerve fibers. Changes in the pattern of innervation can
be assessed using quantitative outcome measures such as
Fig. 15.8 Fungal keratitis. Fusarium imaged using the HRT III confocal
microscope. Horizontal field width = 300 µm. (Courtesy of Dr Brasnu
and Dr Baudouin, Paris, France, and courtesy of Heidelberg
Engineering, Inc. All rights reserved.)
 CHAPTER 15
Confocal Microscopy

A B C

D E F

Fig. 15.9 Confocal microscopy and quantification of morphological parameters of sub-basal nerves. Initial images of sub-basal nerve plexus in the
central cornea in a healthy volunteer (A) with corneal sensation 60 mm and diabetic patient (D) with corneal sensation 40 mm and NDS = 8 (image
size: 400 × 400 µm). B and E represent the results of segmentation from the corresponding images in control and diabetic subjects, respectively.
C and F show traces of the geometry of the sub-basal nerve plexus in a final surface reconstruction. Total fiber length of 4706 and 545.4 µm, nerve
fiber density 0.034 mm/mm2 and 0.004 mm/mm2, and single nerve fiber count 68 and 3 were measured in control subject and diabetic patient,
respectively. (From Zhivov A, Winter K, Hovakimyan M, et al. Imaging and quantification of subbasal nerve plexus in healthy volunteers and diabetic
patients with or without retinopathy. PLoS ONE 2013;8:e52157.)

nerve density, length, branching, and tortuosity.85–87 Using

these techniques, significant differences have been identified
between normal and diabetic subjects (Fig. 15.9), which may
precede the development of diabetic retinopathy.85,86,88
One limitation of corneal confocal imaging of the sub-
basal nerve plexus is that it has a limited field of view. Recent
studies have demonstrated that collecting several single
(nonoverlapping) confocal images is sufficient for detecting
differences in morphological parameters between healthy
and diseased states.87,89 However, in order to map temporal
changes in the overall organization and nerve branching
patterns over large areas of the cornea, the generation of
image montages is required. Both manual and automated
approaches to image registration and stitching have been Fig. 15.10 A montage of the human sub-basal nerve plexus
used to generate wide field montages of sub-basal nerves constructed from 32 HRT–RCM images. A vortex or whorl-like pattern
using the HRT-RCM (Fig. 15.10).90–93 More advanced image of the sub-basal nerve plexus in the infero-central cornea of the subject
acquisition, processing, and reconstruction techniques are is observed. Scale bar, 200 µm. (From Patel DV and McGhee NJ. Fully
also under development that may reduce patient examina- automated montaging of laser scanning in vivo confocal microscopy
tion time, remove motion artifacts, and improve detection images of the human corneal subbasal nerve plexus. Invest Ophthalmol
of sub-basal nerves that are normally obscured by ridges Vis Sci 2005;46:4485–8, Copyright, Association for Research in Vision
induced by applanation pressure, thereby allowing more and Ophthalmology.)
complete reconstructions.93,94

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 PART ii Examining and Imaging the Cornea and External Eye

Section 3 Imaging Techniques of the Cornea

Advanced Technology: Second Harmonic

Generation Imaging of Corneal
Collagen Architecture
The application of lasers to biology and medicine, while
not new, has been greatly expanded by the development
of ultrafast, femtosecond lasers that enable the focusing
of very high-intensity light within small optical volumes
to generate nonlinear optical effects including two photon
excited fluorescence (TPEF), second harmonic generated
signals (SHG), and laser-induced optical break down (LIOB).95
SHG has particular relevance to studying the structure of the
cornea, since collagen fibrils generate strong SHG signals
that can be imaged using optical microscopic techniques.96
To generate an SHG signal, a material is excited by the simul-
taneous absorption of two photons of light of the same
frequency and then the material emits a single photon of
light that is double the frequency or half the wavelength of
the excitation light. The ability to generate SHG signals is
limited to materials that are highly ordered and non-
centrosymmetric, such as collagen.97 Hochheimer, in 1982,
was the first to show that SHG signals could be generated
from the rabbit cornea.98 Past studies have also shown that
Fig. 15.11 Combined forwardscattered (cyan) and backscattered
imaging of SHG signals can be used to establish collagen
(magenta) image of SHG signal from the anterior human corneal stroma.
fibril orientation, as well as study the three-dimensional col- Collagen fibril organization shows a highly interwoven pattern in the
lagen organization.99,100 anterior cornea. (From Morishige N, Wahlert AJ, Kenney MC, et al.
Second harmonic imaging microscopy of normal human and
Human corneal stromal collagen organization keratoconus cornea. Invest Ophthalmol Vis Sci 2007;48:1087–94,
Copyright, Association for Research in Vision and Ophthalmology.)
Imaging of forward scattered SHG signals from normal
human cornea identifies distinct fiber-like structures, approx-
imately 1 µm in diameter, that vary in length and orientation
depending upon the depth within the cornea (Fig. 15.11).
Viewing of sequential images taken deeper within the cornea
shows that these short collagen lamellae extend continu-
ously deeper into the stroma, indicating that the short seg-
ments represented longer collagen fibers that run in and out A
of the plane of focus. This pattern indicates that the anterior
collagen is organized into a highly interwoven lamellar struc-
ture with many lamellae running in a transverse, anterior–
posterior direction and not parallel to the corneal surface.
Deeper within the cornea, collagen bundles become wider,
but continue to pass through multiple optical planes, sug-
gesting that lamellae continued to be highly interwoven. In
the posterior cornea, large numbers of fibers can be detected
that are grouped together into orthogonally arranged lamel-
lae that run parallel to the corneal surface and show markedly
less interweaving than observed in the other regions. B
To characterize the organization of collagen within the
stroma more precisely, high resolution macroscopy (HRMac) Fig. 15.12 Sample HRMac image. (A) Single-plane, zoomed-out
has been used to generate 3-D reconstructions of the indi- HRMac image of a full-diameter corneal cross-section along the vertical
vidual collagen lamellae detected by SHG imaging.101 A meridian. The full scan is comprised of 60 such planes and is made up
typical HRMac image of a human corneal cross-section is of over 80 000 individual images with a total size of 25000 × 6500 pixels
shown in Figure 15.12. This particular sample had a corneal per plane, shown here at a resolution of 3 µm / pixel. (B) Shows the
anterior central cornea at full resolution (0.44 µm / pixel). Note the
arc length of approximately 12 mm. The original HRMac
presence of the ALL (white arrow) and the insertion of collagen fibers
scan was 25 000 by 9000 pixels for each of the 60 planes at (black arrowheads). (Adapted from Winkler M, Chai D, Kriling S, et al.
a resolution of 0.44 µm/pixel and was made up of over Nonlinear optical macroscopic assessment of 3-D corneal collagen
80 000 individual images. Figure 15.12B shows a view of the organization and axial biomechanics. Invest Ophthalmol Vis Sci
central portion of the cornea at full resolution (0.44 µm/ 2011;52:8818–27, Copyright, Association for Research in Vision and
pixel), revealing complex interactions between collagen Ophthalmology.)

188
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B Bowman’s layer that may contribute to the anterior corneal
Fig. 15.13 3-D reconstructions of collagen lamellae at different depths
from Bowman’s layer. (A) Section from a single HRMac plane of the
central cornea overlaid with representative segmented lamellae at
different depths. (B) This panel shows the segmented lamellae
overlaying the single HRMac plane at reduced opacity. (Adapted from
Winkler M, Chai D, Kriling S, et al. Nonlinear optical macroscopic
assessment of 3-D corneal collagen organization and axial
biomechanics. Invest Ophthalmol Vis Sci. 2011;52:8818–27, Copyright,
Association for Research in Vision and Ophthalmology.)
lamellae. Surface renderings showed that branching occurred
three-dimensionally, with some lamellae continuing later-
ally after branching, and with others branching in the
anterior-posterior direction. Figure 15.13 shows a 3-D view
of branching collagen lamellae in the anterior, mid, and
posterior corneal stroma that have been segmented from the
other lamellae in those regions. Extraction of the anterior
segmented collagen lamellae (B) shows a markedly higher
degree of branching than the segmented lamellae from the
deeper stroma, whereas those in the posterior part of the
stroma exhibited almost no branching at all.
3-D reconstructions also identified other distinct collagen
fiber structures including “Bow spring”-like lamellae that
originated from the highly intertwined lamellae directly
beneath Bowman’s layer and arced upwards, fusing with the
Bowman’s layer before arcing back down (Fig. 15.14; blue =
Bow spring fibers, yellow = Bowman’s layer). “Bow spring”
lamellae were, therefore, characterized by a near-parabolic
shape, the apex of which appeared fused with Bowman’s
layer. In part, these Bow spring lamellae represent a dual
“suture” lamellae identified earlier by Morishige et al. that
are lost in keratoconus and are thought to provide mechani-
cal stability to the corneal stroma.102 The insertion of colla-
gen lamellae into Bowman’s layer has been previously
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CHAPTER 15
Confocal Microscopy
Fig. 15.14 3-D reconstruction of “Bow spring” fibers (blue) that fuse
with Bowman’s layer (gold). (Adapted from Winkler M, Chai D, Kriling S,
et al. Nonlinear optical macroscopic assessment of 3-D corneal collagen
organization and axial biomechanics. Invest Ophthalmol Vis Sci.
2011;52:8818–27, Copyright, Association for Research in Vision and
Ophthalmology.)
detected by Komai and Ushiki using transmission electron
microscopy.103 Furthermore, as discussed by Bron, the ante-
rior stromal collagen is known to have extensive anterior–
posterior interweaving with occasionally insertion into
mosaic.104 The observations using SHG imaging complement
these earlier observations and point to the extension of col-
lagen lamellae from Bowman’s layer much deeper into the
stroma than formerly appreciated. In this respect, studies by
Muller et al.105 evaluating swollen human corneas have
shown that the anterior 100–120 µm of stroma is resistant
to swelling and maintains the corneal curvature. While it
has been proposed that the interwoven nature of the ante-
rior cornea is responsible for this mechanical property, this
region also corresponds to the region containing transverse
lamellae that extends to a depth of approximately 130 µm.
Therefore, it is likely that the presence of transverse lamellae
that insert into Bowman’s layer explains the rigidity of the
anterior corneal stroma.
Conclusions
This chapter has described the ability of confocal micros-
copy to resolve noninvasively structural and functional
interrelationships, both temporally and spatially, in the
corneas of human patients. Using confocal microscopy, the
cellular details of fundamental biological processes such
as inflammation, wound healing, toxicity, infection, and
disease, which could previously be studied only under static
or isolated conditions, can now be dynamically evaluated
over time and the effectiveness of treatment modalities
determined. The application of femtosecond laser-based
nonlinear optical imaging of SHG signals from collagen is
an important emerging technology which can be used to
study noninvasively the structural organization of the
human cornea with high spatial and lateral resolution. In
the future, incorporation of multiphoton lasers into clinical
confocal instruments may allow imaging of both cellular
biology and interactions with extracellular matrix in human
corneas, opening up a new world of potential applications.
189
 PART ii Examining and Imaging the Cornea and External Eye
Section 3 Imaging Techniques of the Cornea
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