1 GENERAL INSTRUCTIONS

1.1 Preparation for practical work

Preparation for the implementation of practical work is the independent

study by students of the theory on the topic of this work. In each practical work the
necessary theoretical material and a list of control questions serving to determine
the student's readiness to perform the work are presented. In addition, in preparing
for work, the student should also use a lecture summary.
On control questions the student should give short written answers, which he
can use during the interview with the teacher. Consultations are conducted by the
teacher in accordance with his schedule of consultations.
For practical work, the student must present a report prepared in accordance
with the above requirements. A student who has not prepared a report or who has
not been interviewed by a teacher is not allowed to do the work.

1.2 The order of execution of practical work

Execution of each practical work with the formulation of its results takes
two academic hours. Auditor time student spends:
− for an interview with the teacher on the theoretical part of the work;

− familiarization with the description, instructions and operating rules of

instruments and equipment used in operation;
− conducting an experimental study, processing the results of the study;

− registration of the report.

The report of the work must necessarily end with the conclusions that the
student made independently of the basis of the results of the conducted studies.
Immediately after the completion of the practical part of the work, the
student must submit of the done work before the teacher, while showing
knowledge of the material that is set out in his report.

1.3 Preparation of the report on practical work

The report on the work is performed by each student separately on sheets of
the standard format (210 × 297). It is allowed to use sheets from a student's
notebook "in a cage".
The report should contain:
− purpose of work;

− answers to control questions;

 − technical characteristics of equipment and instruments used in the work;

− results of experimental studies and calculations;

− conclusions from the results of the conducted studies.

The title page of the report is executed in accordance with Appendix A.
The technical characteristics of the equipment and instruments used in the
work can be entered in the table or presented in the same way as in the passport for
this equipment. Conclusions on the work should be briefly stated, but contain all
the necessary information and be confirmed by the data.
Diagrams, graphs and tables should be performed in accordance with the
requirements of ESKD and GOST 2.105-95 [1] using software or a pencil with
help a drawing tool. Reports, drawn up not according to the standard, are not
accepted for protection.

1.4 Safety measures

When working with optical non-invasive diagnostic devices, a potential
hazard may be electric shock, as well as optical radiation, especially laser
radiation.
For electrical safety, optical non-invasive diagnostic devices are made in
accordance with GOST 50267.0-92. When operating these devices, the following
safety measures must be observed:
− before switching on the devices and installations in the network, it is
essential to check the serviceability of the power cord and the radiation units;
− don't turn on the appliance if the power cord is damaged;

− switch on the appliance to the 220 V network through the sockets to

which the ground loop is connected;
− don't leave unattended appliances in the mains;

− don't allow any and especially conductive liquids to flow into the
instruments.
By the degree of danger of the generated laser radiation, optical non-
invasive diagnostic devices comply with GOST 12.1.040-83.
Strictly FORBIDDEN:
− direct laser radiation to the eyes and look parallel to the beam;

− direct laser radiation to metallic, mirror surfaces, white paper and

other white surfaces, and to bring into the area of exposure to the shining objects
(rings, clocks, mirrors, etc.) that cause light reflection and increase the possibility
of falling into eyes.
 2 THEORETICAL PART

The problems of studying microcirculatory and tissue systems occupy one of

the leading places in medical practice. The study of tissue metabolism is extremely
important in determining the individual features of the pathogenesis of various
circulatory disorders and in identifying individual patient sensitivity to various
drugs.
Methods of optical non-invasive diagnostics are one of the new, promising
directions for the development of modern diagnostic methods for the blood
microcirculation system.
In medical practice, pulse oximeters are widely used to measure arterial
saturation and pulse rate, laser Doppler flowmeters analyzing the state of
peripheral circulation, optical tissue oximeters measuring the saturation of
oxyhemoglobin in peripheral mixed blood, and others.

1.1 Features of the anatomical structure and functioning of the

microcirculatory bed of the human fingers
Now special attention is paid to the research of blood microcirculation, i.e.
movement of blood and lymph in the microscopic part of the vascular bed, which
is a structural-functional unit of the human cardiovascular system and is called the
microcirculatory bed.
From the point of view of maintaining homeostasis and homeokinesis tissue
in a single integrated organism, it is important to consider microvessels in
conjunction with the tissue and regulatory elements surrounding them, which form
the basis of the microcirculatory-tissue systems of the human body.
The microcirculatory-tissue system is a structural-functional unit of organs;
a complex consisting of a set of specialized cells of the parenchyma, cells and a
noncellular component of connective tissue, blood and lymphatic microvessels,
endings of nerve fibers and integrated into a single system by regulatory
mechanisms [1].
The terms "microcirculation of blood" means exclusively hemo
microcirculation, i.e. movement of blood through microvessels, the internal
diameter of which does not exceed 100 microns.
 So, microhemocirculatory bed is considered as the final segment of the
cardiovascular system, where the processes of gas diffusion and transcapillary
exchange are carried out. The functional state of peripheral vessels is understood to
mean a complex of properties that determines the objective functions of the
microcirculation system in the form of a systemic response to functional tests,
which reflects the degree of integration and adequacy of the functions by the
performed the work.
As is known, microvessels perform a number of functions [2]:
1) redistribution of blood depending on the needs of the organism;
2) providing conditions for the metabolism between blood and tissues;
3) compensation and adaptation of the organism to extreme environmental
conditions.
The following microvessel groups constitute the intraorganic microvascular
bed: [2]
1) arterioles;
2) precapillary arterioles (precapillaries or metarterioles);
3) capillaries;
4) postcapillary venules (postcapillaries);
5) Venules.
Sometimes pre-capillary sphincters, as well as arteriolo-venular
anastomoses, are referred to separate structural units of the microvascular bed.
The totality of the elements of the microvasculature is called the
microcirculatory unit (module).

Figure 3.1 – Schematic structure of the microvasculature

 Specificity of the microcirculatory channel as an object of investigation is
that its architecture is not the same in different organs and tissues, therefore, its
division into structural units is not always possible.
Arterioles and precapillary arterioles, according to their functional purpose,
refer to bringing vessels. capillaries and postcapillary venules refer to exchange
vessels, and postcapillary venules and venules to retractors vessels.
Precapillaries are microvessels with diameters from 7 to 16 microns, which
do not have elastic elements, but possess an automatic mechanism. The peculiarity
of precapillaries is their high sensitivity to chemical regulation.
The term "precapillary sphincter" is understood to mean smooth muscle cells
or a group of cells capable of completely closing the entrance to the capillary. Pre-
capillary sphincters have an increased sensitivity to regulatory factors and they are
in the areas of arterioles division into precapillaries or capillary drainage from
precapillaries. Their main function is the regulation of the nutritional (capillary)
blood flow.
The most important component of the microcirculation system are capillaries
- exchange microvessels with a diameter of 5-7 (up to 20) μm. The capillary wall is
formed by a single layer of endothelial cells [3]. However, more recently, one can
often find the opinion that the capillary wall consists of two membranes: the inner
endothelial and the outer adventitial with the basal membrane located between
them [1], or the internal endothelial and external basal with the Rugee cells
(pericytes) implanted in it, [2]. In the capillaries there aren't smooth muscle cells,
as a result of which they do not contract, and their ability to stretch is small and
determined by the mechanical properties of the surrounding tissues [1]. Capillaries
of the skin, as well as skeletal and smooth muscles, are constructed according to
the somatic type, their endothelium and basal membrane are continuous, the
number of pores is small, therefore for large protein molecules, capillaries of this
type are almost impermeable, while water with dissolved mineral substances is
passed well [ 2].
Excretory microvessels are the third component of the microcirculation
system. They are small venules with a diameter of 15-20 μm, formed by the fusion
of the venous sections of the capillaries. Small venules emerge into larger ones,
creating a complex system with numerous arteriolo-venular anastomoses. This
department of the microcirculatory channel is subject to numerous structural
variations, depending on the function performed by the organ or tissue.
Postcapillary venules are the first component of the capacitive part of the
microcirculatory channel, formed by small vessels 15-20 μm in diameter, which
arise from the fusion of the venous sections of the capillaries. Postcapillary venules
emerge into small (30-50 microns), and then into larger (up to 100 microns)
venules, thereby forming a complex system of diverting microvessels [1]. Along
with the capillaries they are referred to the exchange vessels.
Arteriolo-venular anastomoses (ABA) or shunts are the vessels that connect
the arteriol with the venule, bypassing the capillary channel. These vessels can be
found in the skin, lungs, kidneys, liver, they have smooth muscle cells and, to a
greater extent, compared to other vessels, are provided with receptors and nerve
endings that regulate blood flow. It is known that the skin of the pads (the finger's
surface) of the finger is rich in ABA, vegetative and sensory nerve fibers, and often
this topographic anatomical site of the tissue is used to evaluate the neurovascular
function and the condition of the microcirculation system in general.
Arteriolo-venular anastomoses perform a number of functions, the most
significant of which are:
1) redistribution of blood to the operating body;
2) oxygenation of venous blood;
3) thermoregulation of a given organ or body part;
4) increased blood flow to the heart.
Microcirculation system reacts sensitively to the strong cooling of the skin
by the reflex spasm of its vessels, which is often accompanied by severe pain, but
gradually the vessels expand. It is assumed that this mechanism is due to the action
of nitric oxide on the smooth muscle cells of the walls of the vessels. Figure 3.2
shows the dependence of the functioning of heat transfer mechanisms and local
thermoregulation under different temperature regimes of the environment [4].

Figure 3.2 - Mechanisms of heat transfer and local thermoregulation in the

skin under various temperature regimes of the environment: under cold conditions,
the blood flow in skin microvessels is sharply reduced (a); in conditions of an
elevated temperature, the blood flow in the skin microvessels increases sharply, the
heat transfer increases (b)

2.2 Features of the anatomical structure of the skin

Skin is the outer covering of a people body, which performs various
functions, protecting the body from destructive environmental factors, participating
in the metabolism and release of metabolic products, in thermoregulatory
processes and redistribution reactions of the circulatory system [5]. Skin is a three-
component tissue system formed by the epidermis, dermis and subcutaneous fatty
tissue (hypodermis), which are in morphofunctional unity and consist of multiple
functional-structural elements (Figure 3.3) [6].

1 - papilla of the dermis of the skin; 2 - groove of the skin; 3 - tactile

rollers; 4 - excretory duct of sweat gland; 5 - sensitive (tactile) body; 6 -
epidermis; 7 - vascular and nasal papillary nets; 8 - connective tissue base of
the skin; 9 - tunica propria corii; 10 - vascular network; 11 - subcutaneous
tissue; 12 - sweat gland.
Figure 3.3 - Structural structure of the skin

The epidermis covers the surface of the skin and is a border tissue with
pronounced barrier functions. The relief and thickness of the epidermis are
different. In the epidermis there is a constant movement of cells and their change.
Germ cells eventually undergo a series of structural and biochemical changes,
eventually turning into horn cells, which in the process of life are constantly torn
from the surface of the skin. The epidermis deepens into the dermis in the form of
epidermal cords.
Dermis - a deep layer or corium, consisting mainly of papillary and reticular
layers, as well as sweat and sebaceous glands. The conditional boundary of the
dermis is the horizontal-branched network of blood vessels and small capillaries.
There are hair bags in the dermis. From the mesh layer of the dermis, in the form
of strands, collagen beams penetrate into the subcutaneous tissue, forming a wide-
loop network, the loops of which are filled with a loose connective tissue
containing many fat cells.
Accumulations of fat cells form a fat deposit. In different areas, fatty tissue
(hypodermis) has different sizes, and in some cases it is absent [7]. Blood vessels
form an expanded volumetric network in the skin, providing good blood supply.
 It has been established that the physiological state of the skin is very
informative from the point of view of the general state of the organism, therefore
the diagnostics of the optical and thermophysical properties of the skin is the most
important procedure.
2.3 Blood physiology
The total amount of blood in the body of an adult is an average of 6-8% of
body weight, which corresponds to 5-6 liters. Blood consists of a liquid part
(plasma) and contain the uniform elements: erythrocytes (red blood cells), white
blood cells (white blood cells) and platelets (blood plates). The proportion of
shaped elements is 40-45%, and the share of plasma - 55-60% of the blood
volume. This ratio is called the hematocrit ratio, or hematocrit number. Often
under the hematocrit number is understood only the volume of blood, which falls
on the share of the formed elements [2].
The main function of erythrocytes is respiratory - the transfer of oxygen
from the alveoli of the lungs to the tissues and carbon dioxide from the tissues to
the lungs. Erythrocytes contain hemoglobin, a respiratory pigment of red color.
The following forms of hemoglobin are physiological:
1) oxyhemoglobin (HbO2) - a hemoglobin compound with oxygen, which is
formed mainly in the arterial blood and gives it a scarlet color, and oxygen binds to
the iron atom through a coordination link;
2) reduced hemoglobin or deoxyhemoglobin (HbH) - hemoglobin, which
gave oxygen to tissues;
3) carbhemoglobin (HbCO2) is a hemoglobin compound with carbon
dioxide, which is formed mainly in venous blood, which, therefore, acquires a dark
cherry color.
Pathological forms of hemoglobin include:
1) carboxyhemoglobin (HbCO), which is formed by carbon monoxide
poisoning (CO). In this case, hemoglobin loses the ability to attach oxygen;
2) methemoglobin (HbMet) - is formed by the action of nitrites, nitrates and
certain drugs, resulting in the transition of ferrous iron to trivalent to form
methaemoglobin.

2.4 Optical properties of skin and blood

Skin is an optically heterogeneous scattering medium with absorption. In
scattering biological media, the processes of interaction with laser radiation have
some peculiarities (Figure 3.4).
 Figure 3.4 – Scheme of interaction of optical radiation with skin

The average index of refraction of the skin is greater than that of air. At the
skin-air interface, a portion of the optical radiation flux is reflected (Fresnel
reflection), and the remainder penetrates into the biotissue. Due to multiple
scattering and absorption, the laser beam broadens and decays as it propagates into
the tissue. Volumetric scattering causes the propagation of a significant fraction of
the radiation in the opposite direction (backscattering). The absorbed light is
converted into heat, re-emitted in the form of fluorescence or phosphorescence,
and is also spent on photobiochemical reactions.
Optical properties of biological tissue are determined by the structure of the
tissue and its state: the physiological state, the level of hydration, homogeneity,
species variability, etc. [8].
The optical characteristics of each layer of the skin are determined by these
or other chromophores (Figure 3.5), for example, the optical characteristics of the
epidermis are considered equal to the properties of melanin.

Figure 3.5 – Chromophores that determine optical properties

different layers of skin

The pass part of the optical radiation stream enters the dermis, where it is absorbed
predominantly by hemoglobin of various forms present in the surface layer of the dermis. The
remaining radiation diffusely reflects from the collagen present in the rest of the dermis. In the
opposite direction, the reflected flux of optical radiation passes through the layers of hemoglobin
and melanin, partially absorbed. The scattering coefficients of the epidermis and the dermis
differ, but these differences are insignificant. The optical properties of the hypoderm are
determined by melanin.
The absorption spectra of light energy by the main chromophores of the skin
are shown in Figure 3.6.
The reflective properties of the epidermis have a strong dependence on the
type of human skin. Figure 3.7 shows an example of the effect of melanin on the
value of the relative reflection coefficient from the human skin of a European type
and an African American. These differences should be taken into account in
studies to reduce the methodological error of measurement.

Figure 3.6 – Spectra of absorption of light energy the main chromophores of

the skin

Figure 3.7 - Relative skin reflection coefficients European and African-American

 When describing the effects occurring in tissues under the influence of
optical radiation, water absorption plays an important role, since it is the main
component of most tissues. In the ultraviolet, visible and near infrared spectrum
wavelength ranges, the water absorption coefficient is very small. In these areas,
the absorption of tissue is determined by the pigment absorption spectra, in
particular for the skin – by the absorption spectra of melanin and blood
(hemoglobin and oxyhemoglobin).
Absorption of the optical radiation flux by blood is mainly determined by its
absorption by water, hemoglobin, and oxyhemoglobin [9]. Oxyhemoglobin,
hemoglobin, and some other compounds and derivatives of hemoglobin give
characteristic absorption bands of the spectrum rays. Thus, by passing a beam of
light through a solution of oxyhemoglobin, two characteristic absorption bands can
be observed in the yellow-green part of the spectrum. For rebuilt hemoglobin, one
broad absorption band in the yellow-green part of the spectrum is characteristic.
The oxyhemoglobin differs somewhat in color from hemoglobin, therefore
the arterial blood containing oxyhemoglobin has a bright scarlet color, moreover,
the brighter, the more completely it became saturated with oxygen. Venous blood
containing a large amount of rebuilt hemoglobin has a dark cherry color. At
wavelengths of 548, 568, 587 and 805 nm, the absorption values of the optical
radiation flux by hemoglobin and oxyhemoglobin are equal. These wavelengths are
called isobystic points.
Whole blood absorbs optical radiation more than hemolyzed, because in it
hemoglobin is in the red blood cells. The increase in the absorption of optical
radiation at short wavelengths of optical radiation is associated with the effect of
scattering on erythrocytes.
As mentioned above, when laser radiation interacts with the biotope, the
absorbed light is re-emitted in the form of fluorescence. Biotissue contains a large
number of different natural fluorophores that have different spectral regions of
absorption and fluorescence, different quantum fluorescence exits, different
fluorescence decay times (Figure 3.8).
Some fluorophores have close and overlapping absorption and fluorescence
regions, as a result of which fluorescence emission from the tissue has a complex
spectral composition.
Quantitative evaluation of the optical parameters of the skin makes it
possible to obtain objective information about the content and spatial distribution
of various biological components in it and successfully use it to diagnose various
skin diseases, study the effects of factors (chemical, UV radiation, temperature,
etc.) effectiveness of treatment and other purposes.
 Figure 3.8 – Absorption spectra (a) and fluorescence (b) by basic
fluorophores of biotissue

Optical non-invasive diagnostics presupposes the use of optical (including

laser) radiation for intravital probing of tissues and organs of the patient in order to
obtain light of information on optical parameters of biological tissues and on
reflected (scattered, past through tissue, reradiated in the form of fluorescence,
etc.) allows on its basis to carry out diagnostics of the biochemical composition
and anatomical (morphological) structure of the examined area of the soft tissues
of the patient's body.