COMENIUS UNIVERSITY IN BRATISLAVA

JESSENIUS FACULTY OF MEDICINE IN MARTIN

TEXTBOOK
MEDICAL BIOPHYSICS

UNIVERSITY
WITH PRACTICAL COURSE

JAKUB MÍŠEK, MARCEL VETERNÍK, JÁN JAKUŠ, ET AL.

MARTIN, 2022
 COMENIUS UNIVERSITY IN BRATISLAVA
JESSENIUS FACULTY OF MEDICINE IN MARTIN

MEDICAL BIOPHYSICS
WITH PRACTICAL COURSE

JAKUB MÍŠEK, MARCEL VETERNÍK, JÁN JAKUŠ, ET AL.

MARTIN, 2022
Authors: © Ing. Jakub Míšek, PhD.
Ing. Marcel Veterník, PhD.
prof. MUDr. Ján Jakuš, DrSc.
prof. RNDr. Ivan Poliaček, PhD.
doc. RNDr. Michal Šimera, PhD.
Mgr. Nadežda Višňovcová, PhD.
Ing. Lukáš Martvoň, PhD.

Reviewers: prof. MUDr. Jana Plevková, PhD.

prof. Ing. Ladislav Janoušek, PhD.

Publisher: Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava,

Martin, 2022. 2nd revised, updated and extended (English) edition.

1st (Slovak) edition: Michal Šimera, Ján Jakuš, Ivan Poliaček a kol. Vybrané
kapitoly z lekárskej biofyziky s praktickými úlohami: vysokoškolské skriptá.
Martin, JLF UK, 2018. ISBN 978-80-8187-056-9.

Printing and reproduction: DOLIS GOEN s.r.o., Bratislava

Language corrections: MUDr. Tomáš Buday, PhD.

Ing. Lukáš Martvoň, PhD.

The authors are responsible for the content of the texts. All rights reserved. No part may be
reproduced without the permission of the publisher or authors.

This publication was supported by the project KEGA 057UK-4/2021.

ISBN 978-80-8187-129-0
EAN 9788081871290
Content

Preface................................................................................................................................... 1
Instructions for the practical sessions ........................................................................... 2
Safety requirements ...................................................................................................... 3
1 Theory of measurement and statistics ............................................................................ 4
Mass.............................................................................................................................. 6
Determination of mass by means of electric scales ............................................ 6
Volume ......................................................................................................................... 7
Concentration ............................................................................................................... 8
Volume measurement .......................................................................................... 8
Measurement of bone density by direct method .................................................. 9
Blood density ......................................................................................................... 10
Measurement of blood density by copper-sulfate method................................. 11
Measurement of density of liquids by means by pycnometer ............................ 12
Statistical data processing .......................................................................................... 13
Statistical data processing ................................................................................ 16
2 Properties of the liquids ................................................................................................. 17
Viscosity ..................................................................................................................... 17
Determination of the viscosity of an unknown liquid........................................ 21
Surface tension ........................................................................................................... 22
Methods of the surface tension measurement ........................................................ 25
Measurement of surface tension of an unknown liquid .................................... 27
3 Biophysics of a cell and cellular membrane ................................................................. 28
Cellular membrane ..................................................................................................... 28
Types of ion channels ............................................................................................ 29
Passive transports ....................................................................................................... 31
Filtration ................................................................................................................ 31
Simple diffusion .................................................................................................... 32
Diffusion through a protein channel ...................................................................... 33
Facilitated diffusion ............................................................................................... 33
Osmosis and osmotic pressure ............................................................................... 34
Osmotic resistance of erythrocytes ................................................................... 37
Active transports......................................................................................................... 39
4 Electric properties of the cells ........................................................................................ 42
Membrane potential .................................................................................................... 42
Action potential and process of excitability ............................................................... 43
Nervous system ...................................................................................................... 46
Synaptic transmission and summation of postsynaptic potentials ......................... 50
Neuro-muscular junction ....................................................................................... 53
Electroneurogram and electromyogram evaluation ............................................... 54
Rheobase and chronaxie ........................................................................................ 56
Calculation of the frequency of the action potentials ....................................... 57
EMG of the m. biceps brachii ........................................................................... 58
Determination of rheobase and chronaxie ........................................................ 59
Measurement of the response of the median nerve ........................................... 60
5 Basics of thermodynamics .............................................................................................. 61
Thermodynamic laws ................................................................................................. 61
Thermodynamics of the living systems ...................................................................... 62
Transformation and accumulation of energy in living systems .................................. 64
Measurement of the organism's energy expenditure .................................................. 66
Measurement of cooling power, calculation of DLH ........................................ 68
The exchange of the heat, thermoregulation .............................................................. 69
Conduction ............................................................................................................. 70
Convection ............................................................................................................. 70
Radiation ................................................................................................................ 70
Evaporation ............................................................................................................ 70
Hypothermia, hyperthermia ........................................................................................ 72
Hypothermia .......................................................................................................... 72
Hyperthermia ......................................................................................................... 73
Temperature measurement ......................................................................................... 74
Thermography ............................................................................................................ 75
Measurement of body temperature by maximal and digital thermometer ........ 78
Measurement of surface temperature by thermocamera .................................. 78
Air humidity ............................................................................................................... 79
Measurement of humidity ...................................................................................... 80
Measurement of air humidity, determination of dew point ............................... 81
6 Respiration ....................................................................................................................... 83
 Biophysics of breathing .............................................................................................. 85
Parallelogram ......................................................................................................... 87
Hering's model of breathing .................................................................................. 88
Lung volumes and capacities ................................................................................. 90
Spirometry ............................................................................................................. 91
Demonstration of intercostal muscle function - parallelogram ........................ 93
Hering's model of breathing ............................................................................. 94
Spirometry - measuring the vital capacity of the lungs .................................... 94
7 Biophysics of heart and circulation ............................................................................... 95
Mechanic work of heart .............................................................................................. 95
Cardiac cycle ......................................................................................................... 95
Mechanics of blood flow ....................................................................................... 98
Mechanic properties of the vessels ........................................................................ 99
Model of blood vessel elasticity .......................................................................... 100
Model of blood vessel elasticity ...................................................................... 101
Blood pressure .......................................................................................................... 102
Blood pressure measurement ............................................................................... 102
Palpation method ................................................................................................. 103
Auscultation method ............................................................................................ 103
Principles of blood pressure measurement .......................................................... 105
Blood pressure measurement .......................................................................... 107
8 Light and it’s properties ............................................................................................... 108
Wave parameters ................................................................................................. 108
Reflection and refraction of light......................................................................... 110
Refractometry ........................................................................................................... 112
Determination of the saccharose concentration using refractometer ............ 113
Spectrophotometry ................................................................................................... 115
Absorption and emission spectrum...................................................................... 116
Determination of absorption spectrum of hemoglobin ................................... 118
9 Sound and ultrasound .................................................................................................. 120
Properties of sound ................................................................................................... 120
Ultrasonic diagnostic methods............................................................................. 121
Types of ultrasonographic views ......................................................................... 123
Measuring the speed of a blood flow in the arteries using Doppler ............... 125
 Tissue perfusion examination using the plethysmographic probe .................. 127
Measurement of tracheal diameter and volumetric blood flow ...................... 128
Effects of sound ........................................................................................................ 129
Infrasound ............................................................................................................ 129
Audible sound ...................................................................................................... 130
Ultrasound ............................................................................................................ 131
10 Optics and microscopy................................................................................................ 133
Imaging using optical systems .................................................................................. 133
Basic terms of geometric optics ........................................................................... 133
Imaging using a thin lens .......................................................................................... 133
Lens equation ....................................................................................................... 135
Magnifying glass ...................................................................................................... 135
Measurement of focal length of a thin converging lens .................................. 137
Measurement of magnification of magnifying glass ....................................... 139
Microscope ............................................................................................................... 140
Imaging using the light microscope.......................................................................... 143
The magnification of a light microscope ............................................................. 143
Phase-contrast microscopy .................................................................................. 146
Fluorescence microscopy ..................................................................................... 148
Special light microscopes .................................................................................... 149
Fiber optics – endoscopy .......................................................................................... 150
Electron microscopy ................................................................................................. 151
Transmission electron microscope....................................................................... 152
Scanning electron microscope ............................................................................. 153
Measurement by light microscope .................................................................. 155
11 Electricity and live organisms .................................................................................... 156
Electric current, voltage, and resistance ................................................................... 156
Passive electric properties ........................................................................................ 157
Conduction of direct current through the tissues ................................................. 157
Measurement of the skin resistance ................................................................ 160
Conduction of alternating current through the tissues ......................................... 162
Measurement of tissue impedance .................................................................. 164
Electric current in living tissue ................................................................................. 166
Electric shocks and injuries ...................................................................................... 167
12 Biophysics of sensory perception – eye, ear .............................................................. 172
Biophysics of an eye................................................................................................. 172
The optical system of an eye ............................................................................... 172
Eye accommodation ............................................................................................ 173
Refractive errors of an eye................................................................................... 173
Astigmatism ......................................................................................................... 175
Scheiner's optometer ............................................................................................ 176
Determination of accommodation width ......................................................... 177
Experiments on Scheiner's optometer.................................................................. 178
Stereopsis ............................................................................................................. 179
Visual acuity ........................................................................................................ 181
Determination of visual acuity ........................................................................ 183
Color vision ......................................................................................................... 184
Color vision deficiency ........................................................................................ 186
Color blindness examination ............................................................................... 187
Examination of color blindness ...................................................................... 189
Biophysics of an ear ................................................................................................. 190
Transmission of sound waves through the ear ..................................................... 190
Determination of hearing threshold ..................................................................... 191
Audiometric examination ................................................................................ 193
13 Electric activity of the heart ....................................................................................... 194
Electrocardiography ................................................................................................. 194
ECG leads ............................................................................................................ 195
ECG measurements ......................................................................................... 199
The electric axis of the heart .................................................................................... 201
Construction of the cardiac electric axis .............................................................. 201
Measurement of cardiac electric axis ............................................................. 203
14 Radioactivity and ionizing radiation ......................................................................... 204
Radioactivity ............................................................................................................ 205
Types of the ionizing radiation ................................................................................. 206
Directly ionizing radiation ................................................................................... 206
Indirectly ionizing radiation ................................................................................ 206
Other types of ionizing radiation ......................................................................... 207
Detection and measurement of ionizing radiation .................................................... 208
 Gas detectors ........................................................................................................ 208
Scintillation detectors .......................................................................................... 210
Semiconductor detectors ...................................................................................... 212
Thermoluminescent detectors .............................................................................. 213
Dosimetric examination of the workplace and personal dosimetry ................ 214
Interaction of alpha, beta, and neutron radiation with matter ................................... 215
Interaction of alpha radiation with matter............................................................ 215
Interaction of beta radiation with matter.............................................................. 215
Interaction of neutrons with matter ...................................................................... 216
Interaction of gamma radiation with matter ............................................................. 217
Measurement of gamma-rays absorption........................................................ 219
Biological effects of ionizing radiation .................................................................... 220
Protection against ionizing radiation ........................................................................ 222
15 Biocybernetics.............................................................................................................. 224
Information and its transmission and processing ..................................................... 224
Types of information ........................................................................................... 224
Biosignals ................................................................................................................. 226
Types of biosignals .............................................................................................. 226
Acquisition and recording of the electric signals ..................................................... 227
The principles of transducers for the recording of non-electric signals ................... 232
Monitoring and telemetry ......................................................................................... 234
Systems – the terminology and their structure ......................................................... 235
The dynamic systems properties............................................................................... 237
The basics of organ control and regulation .............................................................. 240
16 Quantities and units .................................................................................................... 242
SI units ................................................................................................................. 242
Derived SI units ................................................................................................... 243
Prefixes ................................................................................................................ 246
Annex - Tables for practical tasks .................................................................................. 247
Literature .......................................................................................................................... 251
 0 Preface

Message for students

Dear students,
welcome to the practical course Medical Biophysics. Many basic methods of clinical
examination are based on biophysical principles. It is essential that the physician already
during the study should get familiar with the basics of physics, especially related to the
properties of living matter, basic functions of a living organism, and methods of
quantification of normal as well as pathological events. Based on this knowledge, the student
will develop a sense of logical thinking that can successfully determine the optimal and state-
of-art treatment procedures in clinical practice.
We tried to prepare the textbook to be clear, concise, and easy to understand. The
texts are accompanied by a large number of pictures that illustrate the described problem. It
should help you to understand and get familiar with each practical task. Each chapter
contains several tasks with prepared protocols that you will complete during the practical
courses. This publication was published to become an integral part of the subject of Medical
Biophysics, while it also covers some parts of the lectures. However, this book does not
contain all the information you need to successfully handle the subject of medical
biophysics. But we still believe that these texts will help you prepare to better understand
basic theory and successfully pass the final exam. Good luck!

Authors and teachers of Medical Biophysics.

Instructions for the practical sessions

Please, do not write or draw directly to this book! You can find particular
protocols on the JFMED Department of Medical Biophysics website. Those are designed to
be printed without unnecessary notes. Bring printed protocols to be filled in directly during
the practical sessions. Each student has to elaborate on his/her own protocol. Only the
protocol assigned by points and signed by the teacher will be considered as a successful pass
of the practical session.

Printable protocols

100% attendance at practical sessions is required! Students are allowed to

substitute only two practical sessions (missed for a serious reason) during the same week
with another group after agreement by the teacher or during the compensatory week at the
end of the semester. Substitution of more than two practical sessions is possible only with
the permission of the head of the department. The practical sessions during Rector’s and
Dean’s free Days and Slovak national holidays are neither performed, substitution is on a
voluntary basis.
Active attendance at all practical sessions and seminars in Medical Biophysics is
compulsory. Students carrying out a practical session are supposed to be familiar with a
theory and practical topic. If the teacher finds that the student is not ready for the practical
session, the teacher has the right to ask the student for the substitution of the practical
session.
Before leaving, please clean the working bench and prepare the measuring equipment
for the next group.

2
Safety requirements

Basic instructions provide behavioral and safety information to prevent accidents in

the laboratory. The laboratory work takes place in a potentially harmful environment.
Therefore, you should strictly follow the teacher's instructions and safety requirements to
avoid injuries to yourself or your colleagues.
Students are not allowed to eat or drink in the laboratory during the practical sessions.
Instead, students are supposed to wear white coat and overshoes. Do not put anything on
the working bench that you do not need (like bags, clothes, food, etc.). Leave unnecessary
stuff in the cabinets in the hallway (in front of the practical rooms).
Be careful when working with glass equipment since it is one of the most common
sources of injury.
Before starting to work with electric devices, the teacher must give you permission
to do that. Each electric circuit made by the student must be verified by the teacher or
technician. In case of injury, switch off the emergency power button to the OFF position and
report it to the teacher. Never try to touch the person under electric shock! You can be struck
as well. In specific cases (e.g. smelling the smoke from overheated wires), students are not
allowed to find out and repair the error on their own.
When working with material that emits ionizing radiation, follow three basic rules:
• Distance – do not touch the material with the bare hand. Always use some kind
of tweezers. Also, if the emitting material drops on the floor during the
manipulation, never try to catch it with your hand!
• Time – manipulate with the emitter as quickly as possible.
• Shelter – do not leave radioactive material lying on the table. Use appropriate
lead cover.
In case of fire, please act in a way to prevent injury or damage to a greater extent -
use a fire extinguisher as soon as possible. Decisions on further steps will be made by the
teacher or another authorized person. If anyone is wounded, the teacher will immediately
call for an emergency.

Basic safety rules

• Know emergency exit routes, as well as the location of the fire extinguisher and the
emergency power-off button.
• Use equipment only for its designated purpose.
• Minimize all chemical exposures, and avoid skin and eye contact.
• Do not taste or intentionally sniff chemicals!
• Never consume and/or store food or beverages or apply cosmetics in the lab.
• Avoid distracting or startling persons working in the lab - no “horseplay” is tolerated.
• Long hair and loose clothing must be pulled back and secured.
• No cell phone or ear phone usage in the active portion of the laboratories, or during
experimental operations.
• Avoid wearing jewelry in the lab as this can pose multiple safety hazards.

3
 1 1 Theory of measurement and statistics

Quantification is inevitable in every human activity, especially in scientific work. The

numerical expression of the outcomes is the shortest and the most exact way how to
characterize an object. Even in medical practice, there are many parameters that are
expressed numerically. They are mostly determined by measurement (e.g. temperature and
blood pressure, the concentration of substances in blood – hemoglobin, glucose level, etc.).
Actual values are usually compared to a norm – values that are physiologically normal and
indicate no pathological state.
The measurement consists of preparing, execution of the measurement, and verifying
the results. The preparation must clearly state what is going to be measured and how, aids,
instruments, and chemicals must be prepared, the instruments must be properly calibrated
and it must be clear in what form the results will be expressed. During the measurement,
these procedures are performed, with an emphasis on maintaining the correct procedures.
The presence of the measured object (e.g., patient) is important. If the measurement is
performed on the patient, we must first obtain the so-called informed consent. It is also
necessary to maintain the required conditions, e.g. measurement at rest or after exercise, on
an empty stomach, etc. Checking the results is very diverse. From simply “thinking” whether
everything went smoothly and whether the results are meaningful, to performing control
measurements or complex tests.
It is very important to be able to assess the reliability of a numerical expression. In any
case, keep in mind that all measurements are burdened with errors. Considering the
physiological parameters of healthy people, specific values show deviations.

Sources of errors (deviations) are different. From a point of view of their origin, the
errors may be divided into:
• personal – caused by the experimenter (e.g., by the imperfection of his/her sight, or
error in calculation, etc.),
• instrumental – caused by the instruments or apparatus used for the measurement
(electric instability, imperfection of their components, etc.),
• methodical – arising when a wrong or imperfect method is used. Also, when we use
simplified techniques, we are aware of the lower accuracy of the outcomes (e.g.,
neglecting correction to vacuum during the mass measurement).

If we wish to determine the accuracy of an outcome, one single measurement is never

sufficient. When physiological normal parameters are being determined, it is not enough to
measure one single healthy person. That is why repeated measurements are fundamental for
the successful processing of outcomes (we will make sure there has not been any serious
error by repeating even a simple measurement). In medical practice, qualitative data are also
used but the diagnosis is never stated based on a single parameter. We always try to obtain
as much data as possible (although, in this case, we do not speak about repeated
measurements of the same quantity).

4
 In accordance with the nature of the errors present in a set of repeated measurements the
errors (deviations) may be divided into:
• Gross errors occur in particular outcomes that are evidently incorrect (weight of
a patient 66 tons, number of erythrocytes 4 billion in 1 l, etc.). It is easy to see such
an error and the erroneous datapoint is deleted from the set (rarely, if possibly, the
data is corrected – e.g. correct the unit).
• Systematic errors are those that “shift” the measurement in the same way, i.e.
systematically (they are most often caused by a wrong calibration of devices – e.g.
meter is in fact 97 cm, thus, the device will measure more than the reality; laboratory
method destructing red blood cells will cause that fewer erythrocytes will be always
measured, etc.). Such errors cannot be discovered during the statistical processing of
the outcomes. They may be found during examination of the devices or the
measurement technique (or, in medicine, when an examination repeatedly does not
prove the diagnosis confirmed by many other examinations). Such errors are
corrected by a change in the experimenter, the measurement technique, or the
devices.
• Accidental (random) errors cannot be technically eliminated. They are present in
each type of quantitative data (often in the qualitative parameter as well). They are
caused by numerous influences of the surroundings affecting the measurement, by
the imperfection of the devices and technique, but also by natural deviations. For
example, there are no mathematically exact values of the length of a table (at
a molecular level the edges are always uneven, Fig. 1.1). Analogically, the difference
between individuals causes normal physiological data determined in a great number
of people to provide an interval of normal values (e.g. number of erythrocytes in man
is from 4.3 to 5.3 million in μl and woman from 3.8 to 4.8 million in μl). Thus, the
extent of accidental errors is also a measure of accessible accuracy.

Fig. 1.1: Unevenness of knife edge.

5
Mass

A person's weight is constantly changing. The individual consumes and receives

energy, nutrients, excretes, sweats, breathes, etc. so the number of cells in the body is not
constant (they are constantly formed and destroyed by apoptosis). By analogy, the natural
diversity of individuals causes physiological data, which are usually obtained from a large
number of healthy people, to provide a range of values (e.g., the number of erythrocytes in
human blood in men 4.3 to 5.3, in women 3.8 to 4.8 million in mm3 (μl). The magnitude of
random errors is also a measure of the achievable accuracy if the accuracy of the
measurement process is so high that we detect a natural variability. Weight is a quantity that
describes the amount of a substance. The basic unit is kg. Another quantity expressing the
amount is the amount of substance with a basic unit of mol. 1 mol is 6.023 x 1023 particles
(objects). Recalculating weight and mass is easy via the molar mass parameter. For accurate
weight measurement, it is necessary to calculate the vacuum correction or perform the
measurement in a vacuum.

Determination of mass by means of electric scales

Task:
1. Determine the measured parameters in the animal required to prepare an effective dose
of anesthetic.
2. Prepare the solution of anesthesia for injection.

Procedure:
1. Choose an animal and a type of anesthesia.
2. Calculate the dose needed to anesthetize the animal.
3. Prepare the anesthetic solution.
4. Finally, justify the chosen procedure and the use of
laboratory aids with a focus on the weighing procedure,
explain this, and define the terms calibration and zeroing of
scales (Fig. 1.2). Fig. 1.2: Electric scales.

Table of measured values:

quantity value unit

Animal (parameter)

6
Calculations:

Conclusion:

Volume

Volume is the most commonly measured when preparing solutions of a given

concentration. Volume measurement is much less accurate than weight measurement. In
addition, this measurement is significantly affected by thermal expansion. Volume
measurement is performed in measuring cylinders, jars or other measuring containers,
pipettes, etc. (Fig. 1.3). The procedure and accuracy are conditioned by the type of substance,
its quantity (we cannot use a large container to measure a small amount and it is practically
impossible to measure extremely small volumes), temperature, etc.

Conclusion:

Fig. 1.3: Volumetric cylinder, flask, pipettes.

7
Concentration

Concentration represents the amount of a given substance in an amount / the volume

of another substance (in a carrier, most often water). The most important concentrations are
mass concentration and molar concentration. Mass concentration expresses how much of a
given substance is in a volume unit of a mixture, e.g., saline solution contains 9g of salt in 1
liter of the solution – a concentration of 9 g / l. In this case (and in the case of low
concentrations and the water is a solvent) it is possible to use a percentage - 0.9% NaCl
solution. However, the composition of the 0.9% solution is 0.9% NaCl and 99.1% by weight
of the solvent solution. The molar concentration represents the amount of substance
expressed in moles per volume unit of the solution - e.g. physiological concentration 0.154
mol / l NaCl solution (154 mM = 154 millimoles in 1 l). Similarly to the amount of substance
and the weight, the mass and molar concentration is unambiguously convertible with the aid
of the molar mass.

Volume measurement

Task:
1. Describe the parameters, which you use for the preparation of glucose solution.
2. Prepare 13g of 40% glucose solution, describe the procedure and the tools, which you
use during measurement.

Procedure:
1. Calculate and weigh the amount of solid substance
(powder) needed for the preparation of 13 g of 40%
solution and define units.
2. Calculate and measure the amount of water needed for
the preparation of 13 g of 40 % solution and define units.
3. Prepare the solution and substantiate the use of laboratory
aids, and the chosen procedure and explain the procedure
of volume measurement, define the terms volume
expansion, lower meniscus, and parallax error (Fig. 1.4).

Table of measured values: Fig. 1.4: Paralax error in

volume measurement.
quantity value unit
The amount of solid state
H2O volume

8
 Calculations:

Conclusion:

Measurement of bone density by direct method

Density or specific weight expresses the mass per unit volume of a substance. The
units are kg / m3, g / l, etc. The density units are the same as for the mass concentration.

Task:
1.Conclusion:
Calculate the density of the bone sample.

Procedure:
1. Determine the parameters needed for direct measurement of bone density (Fig. 1.5).
2. Make a measurement, describe it and calculate the bone density.
3. Compare the calculated value with the physiological interval of bone density.
4. Substantiate the use of laboratory tools and assess calculated values.
Note. The interval of physiological values is 1700 - 2000 kg/m3.

Bone density
Healthy Osteoporotic
bone bone

Fig. 1.5: Healthy and osteoporotic bone.

9
Table of measured values:

quantity value unit

Bone (1st parameter):
Bone (2nd parameter):

Calculations:

Conclusion:

Blood density

The density of blood depends mainly on its composition, the number of red blood
cells (erythrocytes, Fig. 1.6), and the content of proteins (albumins, globulins, fibrinogen) in
the blood plasma. The physiological value is 1054 to 1062 kg / m3. Due to the use of a
minimal amount of blood, the copper-sulfate method (Phillips and van Slyke) was used to
measure blood density. This method is based on Archimede’s principle and the fact that
CuSO4 solution causes the coagulation of plasma albumins on the surface of the blood drop;
thus, the drop keeps its volume and shape for some period of time. The drop of unknown
blood, being immersed in the CuSO4 solution may: 1. Drop towards the bottom when the
density of the blood is higher than the density of the CuSO4 solution. 2. Move upwards to
Conclusion:
the surface, when the blood density is lower than the density of the CuSO4 solution. 3. Float
within the solution, when both densities are the same (Fig. 1.7).

Fig. 1.6: Erythrocytes.

10
 Measurement of blood density by copper-sulfate method

Task:
1. Determine the blood density by copper-sulfate method.

Procedure:
1. Describe the procedure for density measurement using the copper-sulfate method and
justify its use (physical laws).
2. Drop a drop of blood into a set of tubes with CuSO4 solutions of various concentrations
and densities.
3. We observe the movement of the drop in the solution for 5-10 s. The specific weight
of the solution in which the drop of blood floats corresponds to the specific weight of
the blood. The density of this solution is measured in the next step using a pycnometer.
4. If the drop did not float in any solution, we will estimate the value at intervals of the
density of adjacent solutions, where the drop slowly decreased and slowly increased.
5. Compare the result with physiological values.

Fig. 1.7: Hovering, floating and immersed object in a liquid.

Calculations:

Conclusion:

11
 Measurement of density of liquids by means by pycnometer

The pycnometer (Fig. 1.8) is a glass vessel with a ground-

glass stopper containing a capillary. Excess liquid flows through
the capillary when the pycnometer is closed. When the surface of
the pycnometer is dry, it contains a precisely defined volume.
From the knowledge of the volume and weight of the substance
(in the pycnometer) we can determine the density by definition.
The accuracy of this method, which is straightforward from a
methodological point of view, is affected by the inaccuracy of
volume determination (e.g. thermal expansion, calibration,
wetting of the vessel surface). The procedure by the comparative
method is more accurate. We will perform weight measurements
with an unknown measured liquid as well as with a known liquid Fig. 1.8: Pycnometer.
- usually water (this actually calibrates the pycnometer).

Task:
1. Determine the specific weight of an unknown liquid.

Procedure:
1. Weigh an empty dry pycnometer.
2. Weigh the pycnometer with the measured liquid. Calculate the weight of the solution.
3. Weigh the pycnometer with the reference liquid (water). Calculate the weight of water.
4. Determine the density of the solution according to:
𝑚CuSO4
𝜌CuSO = 𝜌H
4 2O 𝑚𝐻 𝑂
2

Note: Please note that the density of water at a temperature other than 4 ° C is not exactly 1
kg / l.

Table of measured values:

Tube with unknown solution CuSO4 no.

quantity value unit
Room temperature T=

The weight of an empty pycnometer m0 =

The weight of pycnometer with CuSO4 mS=

The weight of the pycnometer with H2O mW=

The weight of CuSO4 solution mCuSO4=

12
 The weight of H2O mH2O=

The density of distilled water ρH2O=

The density of CuSO4 solution ρCuSO4=

Calculations:

Conclusion:

Statistical data processing

The whole set of particular outcomes and errors is not mentioned in the presentation.
The best (the most probable) outcome and the parameter characterizing the range of the
measured values (e.g. statistical error of the measurement) are presented.
Characteristics of accidental quantities (accidental errors (deviations)) are as follows:
Symmetry – it is evenly probable that a specific value will be higher or smaller than the
mean optimal value.
Concentration – particular values “group” around the optimal value (small errors are much
more probable).
Conclusion:
Existence of maximum – the curve expressing distribution or probability of accidental
quantity, as well as particular measured values, that is called normal or Gauss distribution
(Fig. 1.9) has only one maximum. This maximum lies in the position of the most probable
value which is the arithmetic mean (1) of repeated measures in the set. Instead of the whole
set of measurements, the results are presented by this the best and the most probable value
(with a small number of repetitions the arithmetic mean may not be measured exactly at all).
If the measured values are concentrated around their mean, their variability is small.
If they are, on the contrary, dispersed within a far distance from the mean, then their
variability is high. Thus, variability is often measured by means of deviations of the
measurements from the mean.

13
 P probability
( frequency )
P max

P max  
2

measured value x
x + 3 ( e.g. length )
x -3 arithmetic
average x

Fig. 1.9: Normal distribution of frequency of quantitative values -Gauss curve.

x1 + x 2 + ...+ xn x i =1
i
Arithmetic mean x = = (1)
n n

x1 to xn – values of particular measurements

n – number of measurements

The difference between the specific measured value and the arithmetic mean x – xi is
considered an error of the particular measurement. Statistical error is, for example, the value
 (sigma) that is called the mean quadratic error of a particular measurement or the standard
deviation SD.

1
𝑆𝐷 = √𝑛−1 ∑𝑛𝑖=1(𝑥𝑖 − 𝑥̅ )2 (2)

As it has already been mentioned, it is necessary to express also the scatter of particular
values or the statistical error to make clear how “good” the determined arithmetic mean is
(the quality of the measurement). The qualities of the normal distribution have an implication
that the measured value lies within the interval from x + 3 to x – 3 ( x + 3 SD to x – 3
SD) with the probability of 99.7 % (it is about 67% within the interval from x + SD to x –
SD).
Since we are interested in the quality of the arithmetic mean, we need to calculate and
consider analogic value for the arithmetic mean. This is called the mean quadratic error of
the arithmetic mean  (3), nowadays used as a standard error of the mean (SEM) or standard
error (SE). This datum gives information that the calculated arithmetic mean is within the
interval from x + 3 to x – 3 ( x + 3 SE to x – 3 SE) with the probability of 99.7 % (about
67% within the interval from x + SE to x – SE).

14
 1
𝑆𝐸 = √𝑛(𝑛−1) ∑𝑛𝑖=1(𝑥𝑖 − 𝑥̅ )2 (3)

Thus, the basic processing of outcomes consists of the calculation of the arithmetic
mean and the statistical error of the mean. Then, in the presentation of the results the outcome
is written in the following form: mean  statistical error of the mean (i.e. x   units).
In such a record both the mean and the error are expressed in the units of the measured
quantity. In this meaning it is the absolute error (real interval of the value dispersion in real
units). Sometimes we count and express relative error, i.e. an error that is a proportion to
the measured value and is most often expressed in %. The outcome is written in the following
form: mean  relative error: x units  100  / x%.

The other two “mean” values (in addition to arithmetic average) are used to
characterize the data set, particularly for large data sets and data sets with non-Gauss (non-
normal) distribution. Median - the middle value of the dataset ordered by size. If the number
of values of the set n is odd, then the median is the value that has the sequence number (n
+1) / 2, (e.g. for the set: 1, 3, 3, 6, 7, 8, 9 is the median = 6). If n is even, then the median is
the average of values with serial numbers n/2 and n/2 +1 (e.g., for the set: 1, 3, 3, 6, 7, 8, 9,
10 the median equals 6.5). Modus (mode) is the most often measured value (e.g. for file: 1,
3, 3, 6, 7, 8, 9 the mode equals 3).

There are often situations where it is necessary to compare several measured

quantities. The easiest case is the monitoring of one parameter (e.g. frequency of breathing,
frequency of heart, blood pressure) in control conditions and after some interference (e.g.
after administration of a drug, anesthesia, after physical effort, etc.). It is necessary to
measure both the control values and the values after the interference in several individuals,
process each of the sets, and obtain the means and SEs. The fact whether the determined
change (difference between the arithmetic means) of the monitored parameter has been
caused by our interference is found out by means of statistical tests. The T-test (Student’s
test) is suitable for the comparison of two arithmetic means. The outcome of the T-test
calculation characterizes the size of the superimposition of Gauss curves of normal
distributions for the compared means. If their superimposition (probability that the
difference of means is accidental) is smaller than the selected limit (usually 5%), the change
is considered significant, thus, caused by our interference. It is obvious that in practical
medicine as well as in research we monitor more parameters either in more individuals or
gradually within a time frame (yet in different conditions and after different interferences).
Statistical processing of the medical studies as well as the experiments is, thus, much more
complicated. Different procedures, different statistical tests, and methods of numerical and
graphical computer processing are used.
The correct interpretation of the values is necessary for the evaluation and use of the
outcomes. Interpretation (i.e. how to understand the outcomes) and what follows cannot be
done by a machine and there are no universal procedures.

15
 Statistical data processing

Task:
1. Calculate the arithmetic mean, SD, and SE; determine the median and modus in the
set of values (use one of the quantities: finger length, chest perimeter, heart rate, etc.).

Procedure:
1. Measure the selected parameter for particular students (men or women).
2. Calculate the mean; determine the modus and median.
3. Calculate SD and SE.
4. Write the result in the form of absolute and relative error.

Table of measured values:

Measured quantity:

value unit value unit

Student 1 Student 4

Student 2 Student 5

Student 3 Student 6

Calculations:

Conclusion:

16
Conclusion:
 2 2 Properties of the liquids

Viscosity

Viscosity depends on the internal friction of the liquids due to the existence of cohesive
forces which prevent the relative movement of the particles (the cause of different fluidity).
The liquid inhibits the movement of the object immersed in it. But viscosity also
describes the resistance of the liquid to flow. Simply said, not all the molecules move at the
same speed as the fluid flows (e.g., compare honey and water). Thus, the viscosity resists
the shearing motion. In addition, as liquid flows through the tube, the speed of the layers
flowing in the middle is higher compared to the layers which flow closer to the walls (see
Fig. 2.2 laminar flow).
The frictional force (F) arises between the unequally moving layers (shearing flow) of
liquid molecules due to the interaction of the molecules as shown in the formula:

𝛥𝑣
𝐅=𝑆 𝜂
𝑟
where (S) is the area in m2, (Δv) is the difference in layer velocity in m.s-1 and (r) is the
distance of layers in m. This longitudinal friction force is directly proportional to the size of
the contact surface (S), the difference of the layer velocities (Δv), the dynamic viscosity (η)
(read “eta”), and inversely proportional to the layer distance (r).

The viscosity can be expressed as dynamic and kinematic. The dynamic viscosity η
(usually called just viscosity) represents the longitudinal force acting on 1m 2 (F/S), which
rises between the layers of liquid at a perpendicular velocity gradient Δv/r (see previous
formula). The unit of dynamic viscosity is Pa.s (N.s.m-2). The kinematic viscosity (ν) is
defined as a ratio between dynamic viscosity and the density (ρ) of a substance:
η
ν=
ρ

Kinematic viscosity is used to measure the flow resistance under the weight of
gravity. It may be understood as a measure of the magnitudes of viscosity and inertia. The
practical significance lies in the replacement of two letters (η and ρ) with one (ν).
The viscosity of all substances mainly depends on the temperature. As the
temperature of water increases, its viscosity decreases much faster compared to its density
(at 25° C it is almost half the viscosity at 0° C). In the case of Newtonian fluids (e.g., water,
true solutions, and others), the viscosity is constant with respect to the flow rate. For non-
Newtonian fluids (e.g., false solutions, colloids, suspensions, emulsions, blood), the
viscosity depends also on the fluid flow rate.

17
 For example, the blood viscosity also affects the work of the heart when pumping
blood throughout the body. The higher the blood viscosity is, the higher is also the friction
between the blood and the vessel wall. Blood viscosity is affected by several factors such as
temperature, density, composition, or flow rate. At a normal body temperature, the
viscosity of the blood reaches the values 3 - 3.6 mPa.s. These values decrease with increasing
temperature and vice versa (a decrease of 1 °C causes an increase in blood viscosity by 2%).
As the diameter of the vessel decreases, due to the lower flow rate, the viscosity of the blood
decreases.

To measure the viscosity of liquids, these viscometers are utilized:

• Rotary viscometers – directly measure the torque needed to rotate a disk immersed
in the fluid at a known speed (Fig. 2.1).
• Vibrational viscometers – a source of vibrations (resonator) is immersed in the
measured liquid. As the resonator shears in liquid, the energy loss occurs due to its
viscosity. The vibrations that propagate through the liquid are captured by an external
sensor.
• Falling object viscometers – the viscosity is determined from the free fall of the
object (sphere or piston with known size and density) between the marks in the
container through the measured liquid. In medicine, the analogy of such a
measurement is the determination of blood sedimentation by the Fahraeus-
Westergren (FW) method.
• Flow (capillary) viscometers – the viscosity of the measured liquid is determined
from the time in which the liquid drains through a capillary from the upper to lower
reservoir. Capillary viscometers use the validity of Poiseuille-Hagen’s law,
according to which the flow volume of a liquid (V) is directly proportional to the
square of the tube radius (r) and the pressure gradient (Δp) and indirectly proportional
to the liquid viscosity (η) and the length (l) of the tube:

π ⋅ 𝑟4
𝑉= ∆𝑝
8⋅𝜂⋅𝑙

a) b)
Rotor
Chamber filled
with liquid
Disc

Liquid

Resonator

18
 c) 1 d)
2 3

Falling Z1

ball Z2

Z2
Liquid

Fig. 2.1: Schematic representation of different viscometers: a) rotary viscometer, b) vibrational viscometer,
c) falling object viscometer d) capillary Ubbelohde viscometer.

Fluid dynamics

Fluid flow, either the laminar (turbulence is not present, it is considered ordered or
arranged, sometimes called “sheet-like” flow) or the turbulent (characterized by swirls and
eddies, is considered chaotic), depends on the diameter of the tube (d), on the mean flow
velocity (v) and its physical properties such as density (ρ) and viscosity (η). The
dimensionless quantity expressing the physical characteristics of the flow is called the
Reynolds number and could be defined by the formula:
−
𝐯𝜌𝑑
𝑅𝑒 =
𝜂
Reynolds number (Re) expresses the ratio between the inertia of a liquid and its
viscosity. It represents a suitable parameter for determining flow pattern, thus, predicting
whether the fluid flow will be laminar or turbulent (Fig. 2.2). The higher the Reynolds
number, the lower the effect of the frictional forces of the fluid particles on the total
resistance (and higher chance for turbulences).

Fig. 2.2: Schematic representation of basic characteristics of laminar and turbulent flow.

19
 Turbulent flow – Re > 4000, (in vivo for blood
in the vessels > 1100) represents fast flow, low
viscosity, and dominant inertia of the liquid. The shear
layers are irregularly arranged and mix with each
other with a flat flow profile (mixing is highly
efficient). The formation of turbulent flow in the
bloodstream is accompanied by the formation of
sound as a heart echo or a murmur that can be heard
with the ear or a stethoscope.

Transitional flow – 2300 > Re < 4000, refers to

transition from laminar to turbulent flow. The
transitional phase is basically a combination of
laminar and turbulent flow. Regarded to the pipe,
there is turbulence flow in the middle and laminar
flow near the walls.

Laminar flow – Re < 2300, (in vivo for blood

Re < 1100) represents slow flow, high liquid viscosity,
parallel arrangement of shear layers (layers do not
mix), parabolic flow profile (highest flow velocity is
in the middle, lowest along the edges), e.g., blood cells
flow in the middle of the vessels, and plasma near the
vessel walls.
Fig. 2.3: Smoke of a candle - transition
from laminar to turbulent flow.

Examples of Re values: blood flow in the brain Re ≈ 1×102, in the aorta Re ≈ 1×103,
swimming person Re ≈ 4×106, large ship (e.g., RMS Queen Elizabeth II) Re ≈ 5×109.

20
 Determination of the viscosity of an unknown liquid

Task:
1. Calculate the dynamic viscosity of an unknown liquid.
2. In conclusion, compare the viscosity of the unknown liquid with the viscosity of
distilled water.

Procedure:
1. Fill reservoir 2 with distilled water through tube 1 of the viscometer (Fig. 2.4) so that
the level of the water will reach between lines C and D.
2. Close tube 3 with your finger and suck the water with the balloon on tube 4. First,
push the air out of the balloon (keep the balloon pressed), close the hole on the balloon
with your thumb and slowly release the balloon until the water is sucked into the
reservoir 5.
3. Release the balloon (its opening) and then release the tube 3.
4. Using a stopwatch, measure the time t0 for which the distilled water flows between
lines A and B.
5. Rinse the viscometer and repeat the measurement for the unknown liquid.
6. Put your values into the formula and calculate the viscosity of the unknown liquid.
7. In the conclusion, compare the viscosity of the unknown liquid with the viscosity of
distilled water.

For the viscosity of an unknown liquid:

η0 – viscosity of a distilled water
𝜌𝑡 ρ0 - density of a distilled water
𝜂 = 𝜂0
𝜌0 𝑡0 t0 - run-off time of a distilled water
ρ - density of an unknown liquid
t - run-off time of an unknown liquid
Laboratory conditions during measurement:
Room temperature:

Table of recorded values: Value Unit

The density of a distilled water ρ0 =
The viscosity of a distilled water η0 =
Run-off time of a distilled water t0 =
The density of an unknown liquid ρ= Ubbelohde
viscometer.
Run-off time of an unknown liquid t=

Calculation:

Conclusion:

21
Surface tension

The existence of the phase interface (e.g., gas-liquid interface) conditions the
functioning of several biophysically significant phenomena such as surface tension, capillary
action, etc.
The molecules of the liquid act on each other by attractive (cohesive) forces, which
try to reduce the surface of the liquid to a minimum. The magnitude of these forces depends
on the type and number of the molecules, acting only over a very short distance. Attractive
forces in the middle of the volume are evenly distributed in all directions (Fig. 2.5).
Therefore, the final force equals zero. However, due to the asymmetrical distribution of the
attractive forces at the interface (because of the gas-liquid interface, the concentration of the
molecules is much higher in a liquid than in a gas), the resulting force is directed to the
middle of the liquid (for example creation of the water droplet). This phenomenon is known
as surface tension.

Attractive forces on
Air the water surface

Water
droplet Water
molecules

Attractive forces
inside of the water

Fig. 2.5: Schematic representation of cohesive forces acting among the water molecules.

The surface tension σ (sigma) is defined as a force (F) acting parallel to the surface
of a liquid perpendicular to the unit of length (l):
𝐅
𝜎= [N.m-1]
𝑙

We can also define surface tension as the areal density of the surface energy:
𝐸
𝜎= [J.m-2 = N.m-1]
𝑆

22
where (E) is the energy that would be released if the given surface of the liquid (S) were
reduced to zero. In other words, the surface is likely to contract, to reduce, the surface area.
Thus, the work is necessary to expand the area of the liquid's surface (area). Getting back to
units, work (F.s) is basically a constant force (F) that acts on an object in a straight line (s).
The area is in units of squared distance (s2). So, a work per area is basically the same as a
force per length. The purpose of expressing the surface tension by employing the units of
length is to show that if any object is placed at a surface of a liquid, the contact region is
always some kind of curvature (one-dimensional). Thus, the length is finally playing a key
role.

The surface tension is a temperature-dependent quantity. As the temperature

increases, the surface tension decreases, which is a manifestation of the deteriorating of the
cohesive forces in the fluid.
At the point where the gas-liquid interface meets a solid surface, the behavior of the
liquid depends on the mutual ratio of cohesive and adhesive forces. Cohesive forces
represent attractive forces acting between molecules of one type (e.g., water-water).
Adhesion forces are attractive forces acting between molecules of different substances (e.g.,
water – glass).

• If the adhesive forces are greater than the cohesive forces, the liquid wets the
surface of the container (e.g., water). The liquid will rise in the capillary – represented
by capillary elevation (Fig. 2.6 left).

• If the cohesive forces are greater than the adhesive forces, the liquid does not wet
the surface of the container (e.g., mercury). The fluid will decrease in the capillary –
represented by capillary depression (Fig. 2.6 right).

Fig. 2.6: Schematic representation of: left – capillary elevation, right – capillary depression.

23
 As mentioned before, surface tension “tries” to reduce the surface of a liquid to
a minimum. Due to the curvature and the trend to reach the minimal surface, the pressure
inside the liquid increases. The air pressure in a circular bubble (drop) is described by
Laplace's law:

2
p =
r
where (Δp) represents the bubble pressure, (σ) is the surface tension and (r) is the radius of
curvature of the liquid (bubble/drop size).
A substance with the ability to increase the surface tension has the attribute “surface
active”. The surfactants (surface-active agents) are substances with the ability to decrease
surface tension. They are used when cleaning or mixing of different phases is needed (for
example detergents or emulsifiers). Surfactant is an amphiphilic substance (Fig. 2.7) which
contains hydrophilic (water-soluble heads) and also hydrophobic (water-insoluble tails)
parts. It is able to adsorb to the interface between a solution containing water and another
phase (e.g., gas or solid material). Surfactants are often used as the bulk phase in water so
the hydrophobic component is the one pointing outwards.

Hydrophilic Hydrophobic tails

head

Fig. 2.7: Schematic of a surfactant molecule.

Pulmonary surfactant is one of the key substances in preventing the collapse of the
lungs during expiration (atelectasis). This surfactant consists of lipids and proteins creating
a lipoprotein complex. In humans, it is produced by type II alveolar cells. It is adsorbed in a
thin layer of water spread on the inner side of the alveolar walls with the lipid component
pointing outwards. The gas-liquid interface is “willing” to deflate the alveolus to create
minimal volume which leads to an increase in the pressure inside. Not all alveoli have the
same size. As the alveolus gets bigger in size during inspiration, the adsorbed surfactant
molecules become further away from each other. According to Laplace's law, the pressure
is lower as the molecules are further apart and vice versa, the pressure increases with
decreasing distance (radius). In other words, the bigger the alveolus, the lower the pressure
but further the surfactant molecules (small increment from lowering of the surface tension).
The smaller the alveolus, the higher the internal pressure, but surfactant molecules are much
closer together (creating a high increment of the lowering of the surface tension). This
phenomenon ensures that all alveoli will expand at the same rate (same internal pressure).
Without the natural pulmonary surfactants (e.g., in non-matured infants suffering from
infant respiratory distress syndrome), the artificial surfactants must be administrated.

24
 Fig. 2.8: The role of the pulmonary surfactant in regulating surface tension in lung alveoli.

Methods of the surface tension measurement

• capillary elevation method – immerse a narrow tube with a circular cross-section

(capillary) in a vertical position to the liquid poured in a wider container. The liquid
(water) that wets the capillary walls rises to a height h above the level of the liquid in
the wider container - capillary elevation (Fig. 2.9). The weight of the liquid in the
capillary above the liquid level (or below the liquid level for nonwetting liquids) in the
container is held by the force of surface tension acting around the circumference of the
capillary.

2r

h (h 0 )

Fig. 2.9: Capillary elevation method for determination of surface tension.

25
 Then for the surface tension applies:

r - inner radius of a capillary

𝑟ℎ𝜌𝐠
𝑓= h - height of the liquid above the water level
2 ρ - density of a liquid
g - gravitational acceleration

• Stalagmometer (drops weighing method) – the surface tension of an unknown liquid

is determined by a comparative method. It is based on the weighting of a certain
number of drops of an unknown liquid and compared to the known liquid (distilled
water) using the formula to calculate the surface tension of the unknown liquid.

Fig. 2.10: Stalagmometric method.

• Stalagmometer (drops counting method) – a drop falls from the stalagmometer just
when its weight exceeds the force of surface tension. By counting the drops of an
unknown liquid compared to the known liquid (distilled water), the surface tension
could be derived (for details see practical task).

26
 Measurement of surface tension of an unknown liquid

Task:
1. Measure the surface tension of an unknown liquid using a stalagmometer (Fig. 3.9).
2. Explain the dependence of the number of drops on the surface tension of the liquid.

Procedure:
1. Fill the stalagmometer (Fig. 2.11) with distilled water. First push the air out of the
balloon (keeping the balloon compressed), close the hole on the balloon with your
thumb, immerse the end part of the stalagmometer in the water and slowly release the
balloon until the water is sucked above the line Z1.
2. Release the balloon (hole), and pull the stalagmometer out of the water. Count the
number of drops n0 through which the volume of the water drips between the marks
Z1 and Z2.
3. Rinse the viscometer and repeat the measurement for the unknown liquid (n).
4. Put your values into the formula and calculate the surface tension of the unknown
liquid.
5. In the conclusion, compare the surface tension of the unknown liquid with the surface
tension of distilled water.

For the surface tension of an unknown liquid:

f0 - surface tension of distilled water
𝑓0 n0 ρ n0 - number of drops of distilled water
𝑓=
ρ0 n ρ0 - density of distilled water
n - number of drops of unknown liquid
ZZ1
ρ - density of unknown liquid

Laboratory conditions during measurement:

Room temperature:
Z2
Table of recorded values:
Value Unit
The density of a distilled water ρ0 =
The surface tension of distilled water f0 = Fig. 2.11: Stalagmometer

Number of the drops of distilled water n0 =

The density of an unknown liquid ρ=
Number of the drops of unknown liquid n=

Calculation:

Conclusion:

27
 3 3 Biophysics of a cell and cellular membrane

The cell is a basic structural and functional independent unit of living organisms.
There is a total number of 60.000 billion cells with cell size 4-120 µm (10-6 m) in the human
body. The cell represents an open and dynamic system that communicates with the
environment through the exchange of substances, energy, and information. This
communication takes place at the level of the cytoplasmic membrane (Fig. 3.1), which
separates the cell from the extracellular environment.

Cellular membrane

The cytoplasmic (or plasma) membrane is an important part of the cell in terms of its
basic biological and physiological functions.

Membrane functions:
• separates the interior of the cell from the surrounding environment,
• cellular transport – provides the exchange of the substances and energy between
interior and exterior environments,
• semipermeability – membrane is selectively permeable for substances,
• is electrically polarized - the interior of the cell is negatively charged in contrast
with a positively charged extracellular environment,
• the protective function ensures the cell’s size and shape, excitability, immunity,
communication among cells, and reproduction.

Membrane structure:
• the thickness of the membrane is approx. 7 nm,
• a bilayer of phospholipids. Rod-shaped phospholipid molecules consist of a polar
hydrophilic head (phosphates-soluble in water with a positive charge) and two non-
polar hydrophobic chains (hydrocarbon fatty acids insoluble in water),
• cholesterol has the function of "glue". It maintains fluidity (rigidity) and mechanic
stability of a membrane,
• embedded proteins - peripheral proteins are located on the outer side of the
membrane. They perform the function of enzymes and can be easily separated from
the membrane. Integral proteins pass through the entire membrane i.e. are embedded
in a bilayer of phospholipids usually forming the ion channels.

28
Fig. 3.1: Model of a cellular membrane: A - phospholipid molecule, A1 - polar hydrophilic head, A2 - non-
polar hydrophobic chain, B - glycolipid (carbohydrate chains attached to the phospholipid), C -
glycoprotein (carbohydrate chains attached to the protein), D - glycocalyx, E - cholesterol, F - peripheral
protein, G - integral protein, HI - membrane thickness 7 nm

Types of ion channels

Ion channels are created by integral proteins allowing the ions to flow in or out of the
cell. They play a key role in establishing the membrane potentials (either the action potential
or resting membrane potential) or controlling cell volume. There are several ways how the
ion channels respond to the environment - main types of gated channels are: sensitive to
ligand (ligand-gated channels), change in the voltage (voltage-gated channels) or mechanic
force (stress-gated channels), and others (such as sensory neurons sensitive to e.g., light or
temperature). However, each channel state is either open or closed.

Leakage channels

The simplest type of an ion channel, with approximately constant permeability. These
channels are usually always open creating a pore (hole) in a membrane.

Gated channels

The mechanism of channel opening or closing is associated with conformational

changes in the channel-forming protein molecule. The most common gated channels can be
activated chemically (ligand gating), electrically (voltage gating), or mechanically (stress
gating).

29
 Ligand (chemical) gating (Fig. 3.2) is based on the binding of a certain ligand
molecule (e.g., neurotransmitter) to a protein channel, which changes its conformation
resulting in the opening or closing of the channel, e.g., glutamic acid as a ligand in the CNS
is acting to open (activate) channels for sodium and potassium ions.

Receptor Ligand (neurotransmitter)

Chemical
bond

Fig. 3.2: Ligand gating schematics.

In the case of voltage (electric) gating (Fig. 3.3), a change in the protein channel
conformation occurs due to a change in the electrical voltage on the membrane. For example,
during the depolarization phase, the closed sodium channels become open largely changing
the permeability of a membrane for cations.

Change in
polarity

Closed channel Opened channel

Fig. 3.3: Voltage gating schematics.

In stress (mechanic) gating, the channel is forced to open under mechanically induced
stress conditions (changes in physical pressure, thickness, or curvature of the membrane).
They can be found e.g., in the cochlea of the ear, where mechanic sound wave bends the
stereocilia to make the channels open, or in the sensory skin receptors of touch and pressure
(Pacinian corpuscle).

The transport of substances across the cellular membrane can take place by several
mechanisms. According to energy needs, the transport of substances can be divided into
passive and active. In cells, both transport types of substances occur in several forms.

30
Passive transports

Passive transport of substances follows the concentration gradient without energy

consumption in the form of ATP. These include:

• filtration
• simple diffusion
• facilitated diffusion
• diffusion through protein channels
• osmosis

Filtration

The separation of insoluble solids from liquids or gases using a filter medium (e.g., a
biological membrane) is based on different particle sizes, different electric charges, or type
of the molecule. Transport is driven by a pressure gradient e.g., hydrostatic pressure on a
filtration paper (PH=ρ.g.h, i.e. density, gravity, vertical distance) (Fig. 3.4). Another example
is glomerular filtration which takes place in kidneys where nephrons constantly filter the
blood across capillary walls and Bowman's capsule. This filtration is driven by blood
pressure.

Pressure
gradient

Feed
Small
molecule

Filtration layer
Large
molecule

Filtrate

Fig. 3.4: Small particles, which are able to pass through the filtration membrane, become filtrate. In
contrast, large particles remain feed. Filtration is based on pressure gradient.

Filtration depends on:

• the size of the particles (microfiltration, ultrafiltration, nanofiltration)
• the size and number of pores in the filtration layer (membrane)
• the surface charge on the filter layer (positive + or negative - )

31
Simple diffusion

Simple diffusion is a spontaneous movement of the particles following the direction

of the concentration gradient, without binding to membrane proteins and without
consumption of energy in the form of ATP (Fig. 3.5). Fat-soluble substances (e.g., hormones,
ethanol, glycerol, and urea), neutral molecules of H2O, O2 and CO2 penetrate the cell
membrane through the simple diffusion. Diffusion stops when equal concentrations are
reached. Diffusion is not associated with any changes in the volumes.

Concentration
gradient

Dynamic
equilibrium.
End of the
diffusion.

Fig. 3.5: Simple diffusion schematics.

The rule for spontaneous movement (diffusion) is described by 1. Fick's law.

According to this law, the diffusion flux density (J) is directly proportional to the
concentration gradient of the diffusing substance dc/dx through the area (S). The constant
(D) represents the diffusion coefficient (m2.s-1), which expresses the amount of transported
substance in different liquids per second (e.g., 1.10-9 for low molecular weight substances
and 1.10-12 for macromolecules). The diffusion factor is temperature dependent - with
increasing temperature, the diffusion accelerates. Higher temperature represents faster
particle movement. All kinds of diffusion are based on the thermic movement of particles.
Mathematically, Fick's law expresses a differential equation:

𝐝𝐜
𝐉 = −D. 𝑆
𝐝𝐱
Of note: A negative sign adjusts the equation to a positive form since the dc/dx
concentration gradient reaches a negative value - the concentration of the substance
decreases in time.

Diffusion depends on:

• the viscosity of a solvent and particle size of the solute,
• temperature (the higher the temperature of the solution, the faster the diffusion),
• size of diffusion area S (area through which the particles pass during diffusion),
• concentrations,
32
 • distances (diffusion flux decreases with increasing distance).

Diffusion through a protein channel

The transport of larger substances across the biological membrane is ensured through
protein channels. Protein channels are selectively permeable and can be opened or closed
electrically or chemically (gating). Some channels (pores) are permanently open, others can
be opened and closed (channels) depending on the function of the cell or membrane.
Selective permeability means that only certain ions or molecules can pass through the
protein channel. The selective permeability of a channel depends on its size, shape, or the
electric charge of its surface (Fig. 3.6).

Selective permeable protein

channels

Fig. 3.6: Scheme of selective permeability of the membrane based on different shape and size.

Facilitated diffusion

Facilitated diffusion allows the transport of the substances across membranes, which
would not get through by simple diffusion, or only to a very limited extent. Facilitated
diffusion across membranes is mediated by the binding of passing molecules to the protein
transporters, which are formed by specifically structured integral proteins (Fig. 3.7).

Facilitated diffusion:

• represents a passive transport,

• follows the direction of the concentration
gradient,
• is highly selective.

Fig. 3.7: Facilitated diffusion schematics

through protein carrier.

33
Osmosis and osmotic pressure

Osmosis is a type of passive transport mechanism. It occurs at the solution-solvent

boundary (or two solutions of different concentrations) that are separated by a
semipermeable membrane. It represents a spontaneous movement of the solvent molecules
(e.g., water) across a semipermeable membrane from a space (compartment) with lower
osmotic pressure (with a lower concentration of solutes) to a space with higher osmotic
pressure (with a higher concentration of solutes) (Fig. 3.8). Simply said, water “wants” to
dilute more concentrated solution. Osmosis stops when equal concertation on both sides of
the membrane is reached or there is a pressure working against osmotic pressure or if there
is no more solvent (one of the compartments dries). Thus, osmosis is associated with
changes in volume within both compartments.

Fig. 3.8: Schematic representation of osmosis. Water molecules move from lower to higher concentration,
the volume of the solution on the right increases.

Penetration of a solvent into a solution creates an overpressure (osmotic pressure).

The osmotic pressure π can be defined as the overpressure that would have to act on the
solution separated from the solvent by the semipermeable membrane in order to equalize the
levels (to prevent osmosis).
Osmotic pressure (π) is defined by van'Hoff's law:

𝜋 = R . 𝑇. 𝑐. i

where (R) is the gas constant, (T) is the thermodynamic temperature in Kelvins, (c) is
the molar concentration of the solute and (i) is the van'Hoff dissociation factor, which
indicates the number of ions formed during salt dissociation.
Van'Hoff's law describes that the magnitude of the osmotic pressure depends in
particular on the concentration of the solute and the temperature of the solution. The osmotic
activity of substances (magnitude of the osmotic pressure) that dissociate in solution
(electrolytes) is greater compared to substances that do not dissociate (non-electrolytes) at
the same substance concentration.
34
 Osmolarity refers to the molar concentration of all osmotically active particles of a
solution. Osmolality expresses the concentration of osmotically active substances dissolved
in 1 kg of solvent. The comparison of the osmolality of a solution with blood plasma is called
tonicity. Solutions with the same osmolality as blood plasma are called isotonic, solutions
with higher osmolality are hypertonic, andsolutions with lower osmolality are hypotonic.
The osmotic pressure of body fluids or blood corresponds to e.g., a solution with a
concentration of 0.154 mol.l-1 NaCl, also known as saline. Saline solution (9 g of NaCl in
1000 ml of distilled water) is an isotonic (as compared to the blood plasma) solution which
does not contain any other ions (e.g., K+, Ca2+, etc.).
Of note: Saline or physiological solution is a carrier for parenteral administration of
drugs - infusion, irrigation, wound rinsing, etc. The application of saline is not harmful to
the tissues and does not cause a burning or stinging feeling.

Osmotic phenomena in the cell

Changes in osmotic pressure cause size (volume) changes in the cells. However, the
living organism is able to maintain the overall osmotic balance through osmoregulatory
mechanisms. Red blood cells (erythrocytes) maintain their characteristic biconcave shape
only in isotonic solutions in which the erythrocytes do not hemolyze. Hemolysis disrupts the
integrity of the erythrocytes, causing hemoglobin to leave the cells and turn the blood from
an opaque suspension to a clear red solution. In an environment with a lower osmotic
pressure than the blood plasma (hypotonic solution), erythrocytes absorb water due to
osmosis and increase their volume. However, red blood cells hemolyze only at a certain
degree of hypotension of the solution due to disruption of their surface membrane. In a low
degree of hypotension, they survive by creating a ball-shaped cell. On the opposite, in a
hypertonic solution with higher osmotic pressure, erythrocytes lose water. They shrink, and
their membranes may rupture, which results in the leakage of hemoglobin (Fig. 3.9) as well.
Even in an isotonic solution, the water flows occur in and out of the cell at the same rate,
thus keeping the constant volume in both compartments.

Fig. 3.9: Schematic representation of water transport in different concentrations of surrounding solutions.
Left: Hypertonic environment - more dissolved molecules outside than inside. Middle: Isotonic
environment – same number of dissolved molecules outside and inside. Right: Hypotonic environment -
more dissolved molecules inside than outside.

35
 Osmotic resistance of the red blood cells to the hypotonic solution is determined
using a set of hypotonic NaCl solutions with different concentrations. Not all erythrocytes
in healthy individuals are equally resistant to osmotic changes in the surrounding
environment. Some of them will hemolyze even in a small deviation from the isotonic
environment representing the least resistant cells, the so-called minimal osmotic resistance
(e.g., with a damaged membrane, older or pathological erythrocytes). The others are able to
survive without hemolysis even in a strong hypotonic environment representing the most
resistant cells, the so-called maximal osmotic resistance. The difference between the
concentration at which hemolysis of minimally resistant erythrocytes occurred and the
concentration at which hemolysis of even maximally resistant erythrocytes occurred is called
the osmotic resistant width.
Minimally resistant erythrocytes in the healthy blood begin to hemolyze at 75 - 68
mmol.l-1 NaCl concentration (0.44 - 0.40% NaCl) representing the value of minimal osmotic
resistance. All erythrocytes hemolyze at 58 - 51 mmol.l-1 NaCl concentrations (0.34 - 0.30%
NaCl) representing the value of maximal osmotic resistance. Maximal osmotic resistance
subtracted from minimal osmotic resistance indicates osmotic resistance width (e.g., 75 - 58
= 17 mmol.l-1 NaCl; 0.1% NaCl).

36
 Osmotic resistance of erythrocytes

Task:
1. Determine the value of the minimal and maximal osmotic resistance of the red blood
cells from a blood sample and calculate the osmotic resistance width.
2. In the figure, mark the NaCl % and mmol/l concentration for the minimal and
maximal osmotic resistance. Draw the sediment of the erythrocytes and hemolysis with
a red pencil.
3. Compare the obtained values with the physiological standard. Using the table,
determine which model of the blood disease it may represent.

Procedure:
1. Divide into 4 working groups. Each group will obtain a rack with test tubes.
2. Visually estimate the first concentration of the NaCl solution at which the sediment is
seen and the solution is slightly colored - minimal osmotic resistance. Draw the
observation and write the numbers (% and mmol/l) in the table.
3. Visually estimate the first concentration of the NaCl solution at which the sediment is
not seen and the solution is strongly colored - maximal osmotic resistance. Draw the
observation and write the numbers (% and mmol/l) in the table.
4. Calculate the osmotic resistance width (MIN - MAX) and write the value in the table.
5. Finally, compare the measured values with the values of several blood diseases (Table
3.1) and decide which model of the disease it represents.

Minimal osmotic
Maximal osmotic
Disease resistance (%
resistance (% NaCl)
NaCl)
Erythremia 0,40 0,28
Iron deficiency anemia 0,38 0,28
Thalassemia 0,38 0,20
Drug-induced anemia 0,50 0,40
Hepatitis C 0,34 0,22
Physiological value 0,44 0,32
Hereditarian spherocytosis 0,68 0,46
Megaloblastic anemia 0,48 0,36

Tab.3.1: Values of minimal and maximal erythrocyte resistance in some diseases.

37
Laboratory conditions during measurement:
Room temperature: t=

Table of recorded values:

Value Unit Value Unit
Minimal osmotic resistance:

Maximal osmotic resistance:

Osmotic resistance width:

Conclusion:

38
Active transports

Active transport is a movement of substances against the concentration gradient or

the electrochemical gradient that requires the supply of free energy in the form of ATP
(Adenosine triphosphate hydrolysis).

There are two major types of active transport:

• Primary transport
• Secondary transport (Fig. 3.10)

Primary Secondary
Extracellular space
glucose

Intracellular space
glucose

Fig. 3.10: Schematic representation of primary and secondary active transport.

Primary transport

The energy obtained by ATP hydrolysis is used directly (primarily) for ion transport.
For sodium and potassium ions, active transport takes place via a sodium-potassium pump
(Fig. 3.11). The sodium-potassium pump is situated within the cellular membrane in the form
of an enzyme complex (Na+-K+ ATPase) and obtains the necessary energy by converting
ATP to ADP plus free phosphate (converting 1 mol ATP release 33.5 kJ). Na+-K+ pump is
able to transfer sodium and potassium ions through the channel (membrane) in various ratios
e.g., 3:1, 1:2, 2:3 but mainly in 3:2. This pump can be stoped e.g., by tetrodotoxin poisoning
(avoid eating a meal from abdominal parts of fish Fugu).

39
 Fig. 3.11: Schematic representation of sodium-potassium pump.

Other examples of ion pumps:

The proton pump (H+, K+ -ATPase) - allows the formation of hydrochloric acid
(HCl) through the gastric mucosa. It allows ATP-dependent transport of H+ cations from the
parietal cells of the gastric mucosa (into the lumen of the stomach) in exchange for K + ions
entering the cell. It is a type of antiport pump.
Calcium pump (Ca2+ -ATPase) - is found in the endoplasmic reticulum (ER) and in
the plasma membrane of muscles and intestine. It enables ATP-dependent transport of Ca2+
ions from the cytosol into the ER and out of the cell. It is a type of uniport pump.

Secondary transport

It represents a transmission system where the energy is derived secondarily by another

energy-requiring system (cotransports). The energy required to transport one molecule is
obtained from the concentration gradient of another molecule. The concentration gradient of
the second molecule (substance) is usually created by the primary active transport in
advance. For example, glucose molecule “binds” to Na+, then this Na+-glucose complex is
carried through the membrane actively (the glucose-Na+ cotransport), however, following
the Na+ gradient.

• uniport – transport of one substance in one direction (Fig. 3.12)

• symport – cotransport of two substances simultaneously in the same direction, e.g.,
glucose actively transported from the urine together with the sodium ions
• antiport – cotransport of two substances simultaneously, each in the opposite
direction
40
 Fig. 3.12: Schematic of transport proteins (speed of transport is approximately 10 2 – 104 molecules/s).

Other types of active transports

• exocytosis – “cell vomiting” is the process of secretion of larger particles from the
cell by vesicles (e.g., secretion of a neurotransmitter in a chemical synapse),
• endocytosis – “cell eating” is the process of accepting larger particles into the cell
by immersing the particle in the cell membrane, which envelops it and transports it
to the cell in the form of a transport vesicle (e.g., phagocytosis of white blood cells)
(Fig. 3.13).

Fig. 3.13: Endocytosis via an electron microscope.

41
 4 4 Electric properties of the cells

Membrane potential

Changes in membrane permeability are caused by opening (activating) and closing

(deactivating) the ion channels in the membrane following various stimulation (external
stimuli, neurotransmitters - ligands, changes in membrane potential). The stimuli can be
mechanic, electric, chemical (e.g., neurotransmitters), sensory (generator potential), or
synaptic (post-synaptic potentials).
Membrane potential is defined as the difference in electrical potential between the
intracellular and extracellular sides of a plasmatic cellular membrane. Basically, it represents
the voltage of a polarized semipermeable membrane. It equals a summation of equilibrium
potentials of all three major ions (K+, Na+, Cl-). The cause is a different concentration of
these ions on both sides of the membrane. While the outer environment carries a positive
charge, the inner environment possesses a negative charge. This is the reason why we call
this potential negative. For example, the value of resting membrane potential for a nerve cell
is –70 mV, skeletal muscle –70 to – 80 mV, heart muscle –90 mV, and smooth muscle, non-
stable, around –50 mV.

Membrane potential can be altered by:

• changes in ion concentrations – primarily non-physiological
• changes in membrane permeability – physiological

Permeability (relative) of a cell membrane for the ions at rest is K+ : Na+ : Cl- = 100 :
4 : 45 (%). Thus, the resting membrane potential is created by the uneven distribution of
the mentioned ions (Na+, Cl- outside, and K+ inside). Almost all plasma membranes have
the ability to get polarized, usually with negative potential inside (mostly caused by the
negatively charged proteins inside of a cell). The sodium-potassium pump is responsible
for keeping this imbalance to ensure that the correct ion distribution establishes proper
potential, thus, introducing starting point for excitation – action potential. The membrane,
consisting of a phospholipid bilayer, acts as an insulator between the outer and inner
environment.
Goldman's equation determines the magnitude of resting membrane potential
considering the ions that play important role in its formation. In addition to the constants (R,
T, F) it incorporates also the concentrations of diffusible ions ([Na+], [K+], [Cl-]) and their
permeabilities (PNa+, PK+, PCl-). Indeed, the membrane potential is specific at each time and
space by concentration and electric gradients of these “significant” (diffusible) ions:

RT 𝑃𝑁𝑎+ [𝑁𝑎𝑒+ ] + 𝑃𝐾+ [𝐾𝑒+ ] + 𝑃𝐶𝑙− [𝐶𝑙𝑖− ]

𝐸= 𝑙𝑛
F 𝑃𝑁𝑎+ [𝑁𝑎𝑖+ ] + 𝑃𝐾+ [𝐾𝑖+ ] + 𝑃𝐶𝑙− [𝐶𝑙𝑒− ]

42
where E – membrane potential, R – universal gas constant, T – the thermodynamic
temperature in Kelvins, F – Faraday constant, P – permeabilities for given ions, [] – ion
concentrations (in e – extracellular or i – intracellular space).

Action potential and process of excitability

Nerve and muscle tissue can respond to stimuli by excitation. The process of
excitation is a change in the membrane potential of the cells (the potential difference inside
the cell compared to the outside of the cell). Generally, the depolarization (Fig. 4.1)
represents an increase in membrane potential, i.e. the membrane potential becomes
intracellularly less negative. Vice versa, the hyperpolarization (Fig. 4.1) generally
represents a decrease in membrane potential, i.e. the membrane potential becomes
intracellularly more negative.

Fig. 4.1: The resting membrane potential of a neuron is -70 mV. Values higher (less negative intracellular)
represent depolarization (lower polarity) of the membrane, values lower (more negative intracellular)
represent hyperpolarization (higher polarity) of the membrane. Local (gradual) depolarization or
hyperpolarization is continuous (not jump). Its size depends on the strength of the stimulus and thus the
number of open (closed) ion channels (change in membrane permeability). It lasts only several ms.

Depolarizations and hyperpolarizations are gradual on excitable membranes (sensory,

dendritic and somatic, postsynaptic membranes) and their magnitude depends on the stimuli
that caused them. These electric changes propagate by the ion flux to and from the
surrounding environment with a decrement and without repeated stimulus they will extinct
within a few tens of ms. Thus, the resting membrane potential can be recovered. Exciting
membranes (nerve and muscle fibers) respond to a sufficient (above-the-threshold)
stimulus by an action potential.
43
 Excitation, in the form of the action potential, is a form of response (signal, impulse)
by which information is spread over a long distance in the body (along nerve or muscle
fibers). The action potential is described by a very fast, strong phase of depolarization (rising
phase) up to transpolarization (overshoot - spike), immediately followed by repolarization
(falling phase) and terminated by hyperpolarization (undershoot) phase (Fig. 4.2).

Fig. 4.2: A Schematics of the action potential as a form of the cell excitement. B Representative illustration
of a real action potential behavior.

The action potential is triggered by reaching the membrane potential threshold

depolarization. At this membrane potential, the fast voltage-gated sodium channels open
(Fig. 4.3) what increases the membrane permeability for Na+ ions. By the electrochemical
gradient, the influx of sodium ions occurs and the membrane potential reaches positive
values (transpolarization). Thus, the voltage-gated sodium channels are extremely
important in membrane excitation and action potential generation. Their activation and
opening take only a fraction of a ms, however, a closed and inactive state lasts several ms (it

44
is not possible to open the channels in any way, Fig. 4.4). Inactivation of sodium channels,
diffusion of Na+ ions out of the cell and opening of potassium voltage-gated channels
(activated by very low or even opposite membrane potential) leads to a sharp return of
membrane polarization back to negative values - repolarization (Fig. 4.3). The course of
rapid depolarization and repolarization is called a spike and usually lasts about 1-2 ms in the
neuronal cells. The subsequent hyperpolarization phase occurs several ms after the
repolarization due to activation of the potassium channels. K+ channels are then closed (no
further stimulation present) and the membrane potential is likely to return back to resting
membrane potential. Na-K pump (Na-K ATPase) pumping Na+ to extracellular space and
K+ to intracellular space is significantly involved in stabilizing “regular” ion concentrations
throughout the cell.
The action potential is governed by the ALL-or-NOTHING law. There is either the
complete action potential or no action potential at all. In other words, if the threshold level
was reached, the whole action potential (all stages of depolarization, repolarization, and
hyperpolarization) occurs.
Once the sodium channels are open or inactivated, it is not possible to open them again
and fire another spike – the cell is in the absolute refractory period. However, while the
sodium channels begin to recover and also electrochemical gradient starts to be restored,
there is a possibility to induce another action potential, but this “stimulus” (depolarization)
must be strong enough (stronger than that at resting membrane potential). The cell is in the
relative refractory period. The function of refractory periods is to ensure, that action
potential is spread in one direction only (not able to travel backward) and also fulfills a
protective function as it determines the maximum frequency of the spikes (not to get the cell
membrane overexcited or even damaged).

Fig. 4.3: The schematics of the neuronal action potential. 1) resting membrane potential (-70 mV) - action
potential is triggered by an adequately strong stimulus - depolarization (-55mV), 2) closed sodium channels
– reaching transpolarization (+ 30 mV), 3) potassium channels are open, 4) gradual repolarization, 5)
hyperpolarization (-90 mV), 6) gradual return to resting membrane potential by involving of Na-K pumps.

45
 Fig. 4.4: Representation of different states for the ion channel. Closed: ready to be opened. Open: ions are
able to diffuse or penetrate the membrane. Inactivated: impossible to open a channel.

Nervous system

The nervous system is divided into central (brain and medulla oblongata) and
peripheral (nerves and ganglia) parts. It consists of the nerve cells (neurons), including
their afferent and efferent fibers and connections. It performs 3 basic functions: regulation,
coordination, and integration. The regulation provides the management of the body's
functions and their adaptation to immediate needs and demands. Coordination ensures
harmony and synergy between organs and organ systems. Integration guarantees that the
organism behaves as a whole.
The neuron or nerve cell (Fig. 4.5) has 3 basic functional parts. Soma (cell body)
contains DNA, provides the metabolism of the cell, and is a place of integration (summation)
of local changes in membrane potential. Moreover, the neurotransmitters are produced in the
soma. Dendrites (short afferent branches) receive and gather the information (coming in
form of action potential from elsewhere), especially through synaptic connections. The
neurite – axon (long efferent branch) spreads the information from one neuron to another
or from the neuron's body to the synapses connecting the target cells. Between the body and
the axon, there is an initiation segment (axon hillock), where all information from all
dendrites and soma surface are “collected” and the threshold is evaluated. Structurally,
dendrites and soma do not contain fast Na+ voltage-gated channels while initiation segment
and axon do (can produce action potential). Based on the summary potential at the initiation
segment, the action potential could be not initiated (potential below threshold) or initiated
(potential above the threshold) and sent down the axon.

46
 Fig. 4.5: Schematics of the neuron.

Myelin sheath is a lipoprotein complex created by a special type of cell. Myelin in the
peripheral nervous system (PNS) is formed by the Schwann cells and in the central nervous
system (CNS) is formed by the glial cells (oligodendrocytes). Each axon is “wrapped” in the
lipid-rich (fatty) tissue and as one of the basic components of the white matter of the brain
and spinal cord, is the cause of its white color. Axon, as a wire of the nervous system with
the ability to pass electric information (action potential), has an insulating layer (myelin)
around it. However, the myelin does not provide a one-piece cover along the whole axon.
The myelin sheath is divided into longer, non-conductive sections separated from each other
by shorter non-insulated, conductive gaps called nodes of Ranvier (Fig. 4.6).

Fig. 4.6: Myelin sheath “wrapped” around the axon.

47
 Fig. 4.7: Myelinated PNS axon under the electron microscopy showing myelin concentric layers
surrounding the neuronal axon with the cytoplasmatic organs inside.

Information (action potential) generated by the stimulus is transported through the

body along the nerve fiber. This can be achieved either by continuous (unmyelinated nerve
fiber and muscle) or saltatory (myelinated nerve fiber) conduction.
Continuous conduction means that the action potential is gradually created at each
location (segment) of the fiber. It is done by the mechanism of local electric currents. The
action potential at a given membrane place induces the flow of ions into and out of the cell
and surrounding environment (depolarization and repolarization process). When the
threshold level is reached at the neighbor spot on the membrane, the sodium channels are
also open at this place and the action potential is triggered there as well (Fig. 4.8). Thus, the
activation continues and the action potential passes along the fiber. Inactivation of sodium
channels (refractory phase) provides that at each active point (segment) the action potential
does not spread along the fiber in the opposite (antidromic) direction at the same time. The
speed of propagation of the action potentials is about 1 m/s.
Saltatory conduction of the action potential occurs on myelinated nerve fibers. Since
myelin insulates the membrane and does not allow electric changes or ion flux at a given
location, membrane changes occur only in the nodes of Ranvier. The ongoing action
potential in one node will cause ion flux and electric changes in the following node (Fig.
4.9). Thus, the action potential is activated in the nodes only, and the entire section under
the myelin is omitted. The speed of propagation of the action potential can reach up to 120
m/s (the thicker the fiber, the faster the signal).

48
 Fig. 4.8: One-way continuous conduction of an action potential (AP). At time 0, AP is at the 2-mm
position on the axon, the membrane is depolarized (by local currents), the threshold is reached, the sodium
channels are open (purple part) and the rapid depolarization to transpolarization occurs. It is followed by
closure of Na+ channels, opening of K+ channels and repolarization stage (right half of the green part).
Hyperpolarization is than result of still open potassium channels (left half of the green part). Because the
Na+ channels at the 1-mm position are still inactivated (green), they cannot yet be reopened by the small
depolarization. Each region of the membrane is refractory (inactive) for a few milliseconds after an AP has
passed. Thus, the depolarization at the 2-mm site at time 0 triggers AP downstream only; i.e. at 1 ms an AP
is passing the 3-mm position, and at 2 ms, AP is passing the 4-mm position.

49
 Fig. 4.9: Saltatory conduction. The action potential (AP) in Ranvier notch A (intracellular positive electric
charge at a given site) leads to electric currents (intracellular Na + flow from the ongoing AP to the next
notch, extracellularly opposite) and membrane depolarization in the following notch B with AP formation.
Notch C is inactive at the moment.

Synaptic transmission and summation of postsynaptic potentials

The transmission of information from neuron to neuron, from neuron to muscle fiber,
or from neuron to gland cells is called a synapse. Chemical synapse (Fig. 4.10) provides
one-way transmission of action potential from the presynaptic to the postsynaptic part. The
transmission using a mediator requires a certain time (so-called synaptic delay of about 0.5
ms).
A synapse consists of a presynaptic membrane, a synaptic cleft, and a
postsynaptic membrane. The presynaptic part contains vesicles with neuromediator, while
the membrane also contains voltage-gated Ca2+ channels. When the action potential reaches
the synaptic terminal, Ca2+ channels are activated, and calcium ions enter the intracellular
space. The presence of Ca2+ ions in the cell activates the contractile proteins (SNARE protein
complex), which attract the neuromediator vesicles to the presynaptic membrane. The
vesicles fuse with the presynaptic membrane and by exocytosis, the neurotransmitter is
released into the synaptic cleft. The neurotransmitter molecules diffuse to reach the
postsynaptic membrane (mediator molecules activate receptors on the subsynaptic part of
the postsynaptic membrane). This membrane contains ligand-gated ion channels. The more
of these channels are open, the more ions of a given type (according to the type of channels)
enter the intracellular space of a postsynaptic cell. The result is a change in the membrane
potential of the cell close to the subsynaptic membrane (the changes could be expressed by
the Goldman equation), e.g., depolarization occurs when ligand-gated Na+ channels are open
(similarly to an action potential, when voltage-gated Na+ channels are in use). The final
electric event depends on a type of a neurotransmitter (excitatory or inhibitory).
50
 Fig. 4.10: Schematic representation of a chemical synapse:

A) presynaptic part of a cell with a presynaptic membrane,

B) postsynaptic part of the cell with postsynaptic membrane,
1) mitochondria,
2) neurotransmitter vesicle,
3) autoreceptors (suppress neurotransmitter release)
4) synaptic cleft,
5) receptors (ligand gated channels) on the postsynaptic membrane,
6) voltage gated Ca2+ channels,
7) fusion of the vesicles with the presynaptic membrane,
8) neurotransmitter retransmission.

If the molecules of the neurotransmitter interact directly with the receptors, which
act as ion channels, a change in the penetration of ions across the membrane occurs directly
- ionotropic receptors. The neurotransmitter interaction with a receptor protein on the outer
part of the postsynaptic membrane subsequently activates G-protein on the inner side of the
same membrane occurs due to the metabotropic receptors.

According to the method of information transmission, the synapses are divided into:
• electric – bidirectional, fast transmission, direct connection of two neurons (not
prevalent in mammals),
• chemical – one-way, slower, there is a gap between the neurons, through which the
neurons communicate using the neuromediators – ligands called neurotransmitters,
• electro-chemical (mixed) – a combination of electric and chemical information at
one synapse (especially in lower vertebrates),
or according to their effect:
• excitatory – increase the probability of an action potential generation in the
postsynaptic neurons (e.g., neurotransmitters: glutamate and acetylcholine),
• inhibitory – reduce the probability of an action potential generation in postsynaptic
neurons (e.g., neurotransmitters: GABA and glycine) (Fig. 4.11).

An electric synapse provides a direct connection with the neighbor cell utilizing the
gap junctions. Their main difference from the chemical synapse is that they allow the direct
spread of the signals between the cells without the delay (fast transmission) or need for a
mediator. Electric synapses are common in invertebrate and nonmammalian nervous
systems but infrequent in mammals. They are abundant during nervous system development
or in CNS e.g., between glial cells and myelin sheath (Fig. 4.6). They are crucial to the
functioning of the cardiac muscle cells and smooth muscles.

51
 Fig. 4.11: Excitatory mediator (e.g., Glutamate) increases membrane potential on the postsynaptic
membrane – increasing probability to reach the threshold. Contrary, inhibitory mediator (e.g.,
GABA) decreases membrane potential on the postsynaptic membrane – decreasing probability to
reach the threshold.

The change in membrane potential, depolarization, or hyperpolarization of the

postsynaptic membrane, is called the postsynaptic potential - PSP. In the case of
depolarization, the excitatory postsynaptic potential - EPSP (excitatory synapse) is
defined, in the case of hyperpolarization, the inhibitory postsynaptic potential - IPSP
(inhibitory synapse) is expressed. Generally, the postsynaptic potential is a local change of
membrane potential, which spreads within the environment with decrement, i.e., its size
decreases with distance and time.
PSP summation represents the process of combining the multiple simultaneous
inputs (summation in space) or repeated inputs (summation in time) at a given membrane.
The formation of the action potential at the postsynaptic neuron is conditioned by reaching
the threshold (at initiation segment) by the summation of the particular electric events
(electric changes) from the individual synaptic transmission. The action potential is initiated
only if the summation of various EPSPs and IPSPs is sufficient enough to reach the threshold
at the postsynaptic membrane.

The summation of the postsynaptic potentials can be divided into:

• spatial – summation of the PSP from multiple synapses/neurons,
• temporal – summation of the PSP from one synapse firing the impulses repeatedly
in proper time (Fig. 4.12).

The complex process of excitation and inhibition of a postsynaptic cell by synaptic

inputs is called integration. If a constant above-threshold potential (at the initial segment of
the neuron) is maintained, the neuron generates the action potentials and sends them along
the axon with a certain frequency. At the axon hillock of a postsynaptic neuron, the
summation of EPSPs occurs to form an action potential. In order to reach the threshold level
in the axon hillock trigger zone, several EPSPs are needed to summate in proper time, space,
or a combination of both.

52
 Fig. 4.12: Combination of the excitatory impulses (EPSP) and inhibitory impulses (IPSP) could lead to
different behavior of the membrane potential at the subsynaptic membrane. Electric signals can be even
cancelled or summed together to reach the threshold.

Neuro-muscular junction

The nerve fiber that is functionally connected to the effector (muscle, gland) is called
the motoneuron. In a muscle cell (skeletal muscle fiber), the action potential is similar
compared to a nerve fiber. The difference in synaptic transmission from motoneuron to
muscle, compared to neuronal transmission, is that the mediator is always excitatory
(acetylcholine), muscle cell receptors are called nicotinic acetylcholine receptors, and a
single PSP always reaches the threshold and triggers a muscle action potential (EPSP). Since
the activation of a muscle cell comes from only one motoneuron, the summation resulting in
contraction is always temporal. The synapse between the motoneuron and the muscle fiber
is called the neuro-muscular junction (motor endplate) and the EPSP that arises in it is
called end-plate potential.

Fig. 4.13: Muscle contraction curve showing a single muscle twitch (Twitch) resulting from a single action
potential. Incomplete (Unfused) tetanus represents an overlapping of the twitches with the partial
relaxation between action potentials. Complete (Fused) tetanus occurs during high rate of stimulation
representing the maximum strength of a muscle contraction.

53
 An increase in the contraction (strength) of a muscle cell is achieved only by
increasing the frequency of the stimulation initiated by a corresponding motoneuron. All
muscle fibers innervated by a given motoneuron are called the motor unit. The muscle cells
innervated by the same motoneuron within the motor unit will contract at the same time.
Thus, each motor unit must be stimulated by a separate motoneuron. This process offers
regular dosing of the strength of each muscle.

The single stimulus to the muscle cell (motor unit) will cause a single muscle twitch
(Fig. 4.13). With the increasing frequency of the action potentials, the twitches overlap (the
duration of the single twitch is longer than the duration of incoming action potentials) which
leads to temporal summation resulting in either incomplete (unfused) or complete (fused)
tetanus (tetanic contraction). In the case of complete (fused) tetanus, the force exerted by a
muscle fiber is the greatest. The overall muscle strength is controlled and increased by the
involvement of other recruited motor units (involvement of the other motoneurons and
their corresponding muscle fibers).

Electroneurogram and electromyogram evaluation

Generally, single unit activity represents a record of the individual action potentials
of the same amplitude originating from one neuron. The spikes are clearly visible in the
record. Contrary, multipotential represents the spikes within a record that are not clearly
visible (superimposed), multiple with different amplitudes and significant overlap.
Using the single unit activity record, it is possible to measure the intervals between
individual action potentials and thus, the frequency of the bursting. The average frequency
is determined by counting the number of spikes (n) per time unit (T [s]) or by counting the
number of spikes in the burst divided by the duration of the activity:

f (average) = n / T [Hz]

The maximum frequency of discharges is calculated as an inverse of the shortest

inter-peak interval:

f (max.) = 1 / T (min. inter-peak interval) [Hz]

In a multipotential record, we can only evaluate the duration of the whole activity
(burst) and, after postprocessing, its relative intensity.

EMG and ENG

Electromyography (EMG) as a method, measures and evaluates the electric activity

of the muscle. EMG is frequently induced in response to electric stimulation. EMG is
performed using an electromyograph (device) to produce an electromyogram (a record of
54
multipotentials or single muscle unit activity). There are two different methods to obtain
EMG. A metal needle electrode provides more precise information concerning the activity
of individual muscle fibers. For this purpose, a needle electrode is inserted directly into
a muscle and the electric activity is thereby measured. The second method involves the
surface electrodes recording the activity of more muscles together (a multipotential).
When e.g., the biceps muscle is relaxed (resting phase of the muscle), there is almost
no electric activity observable. Compared to the resting phase, during a contraction, electric
potentials are generated. These electric potentials vary with the intensity of the contraction.
The level of electric activity correlates with the number of motor units that are activated.
The experiment shows that the recording of action potentials during the contraction of the
muscle enables the distinction of its flexion and extension movements. The electric activity
(strength) of the biceps muscle is the highest during the flexion of the arm. This is due to the
fact that more force is needed for moving the weight against the gravitational force of the
Earth.

Electroneurography (ENG) is a neurological diagnostic method that determines the

ability of nerves to transmit electric impulses. Electroneurogram (a recording) includes
information on the speed of the action potentials passing the nerve fibers. ENG is usually
used when there is a suspicion of disease or damage to the peripheral nervous system. Such
a method could be used for example to measure the conduction velocity of a median nerve
in the wrist. Morphological failure of this nerve (carpal tunnel syndrome) causes pain,
numbness, and tingling in the hand and arm. Compared to EMG, an electroneurograph (a
device) uses electric impulses to stimulate a neural fiber tissue until it starts to fire the action
potentials. The spikes are then recorded by the surface electrodes with the evaluation of
motor and sensory responses. Even if the stimulating electric impulses are weak, they may
cause discomfort to the examined person.

Fig. 4.14: Left: electromyograph (EMG device). Right: electroneurograph (ENG device).

55
Rheobase and chronaxie
Several diagnostic and therapeutic methods such as electrocardiography (ECG),
EMG, electroretinography (ERG), etc., are based on sensing the electric properties of tissues
(excitement or neuronal responses) and organs (e.g., muscle contraction). The properties of
the tissues can be examined by electric stimulation with a direct current (DC) or low-
frequency electric current (e.g., typical for ENG). The effect of the low-frequency electric
current on a given tissue or cell depends upon its type (DC or AC), intensity of the current
(stimulus strength), direction, etc. Direct current running through the human body remains
below the stimulus threshold (no response occurs) if the current density is approximately 50-
200 µA/cm2. Higher current densities produce a tissue response.
The basic parameters characterizing the activation of neuromuscular activity (tissue
response) are rheobase and chronaxie described by the strength-duration curve: Hoorweg-
Weiss curve (dependence of the intensity I of the rectangular current that causes irritation
with the pulse duration t) (Fig. 4.15). The lowest, threshold, the intensity of the electric
current that will elicit a response (e.g., a nerve fiber) is called rheobase. The rheobase is a
representation of tissue excitability: low values indicate high excitability and vice versa.
The time interval necessary to electrically stimulate a tissue, using the stimulus intensity
twice of the rheobase is called the chronaxie. In healthy muscle tissue, chronaxie usually
lasts 0.3 - 0.5 ms: the higher the value is, the slower a fiber is. In the case of tissue damage
(or reduced nerve fiber conductivity), the Hoorweg-Weiss curve is significantly shifted
upwards and to the right.
In electrodiagnostics, to determine these parameters, a positive pole of the surface
stimulation electrode is placed on the skin at a point that is supplied by the nerve under the
examination. The negative pole of the electrode is placed distally 5 - 10 cm away. Typically,
a rectangular current with an intensity of 0 - 50 mA with a pulse duration of 0.05 to 30 ms
is used.

Fig. 4.15: Comparing rheobase and chronaxie in high speed [1] and low speed [2] nerve fibers.

56
 Calculation of the frequency of the action potentials

Task:
1. Measure the average and the maximum frequency of the spikes in a record of a single
unit activity of a respiratory neuron, a motor unit, or a gallbladder smooth muscle
activity under different conditions.
2. Measure the duration of multipotential nerve or muscle activity.

Procedure:
1. Calculate action potentials (spikes) in the record of a single unit of neuronal activity.

2. Measure the duration of the activity from the first to the last spike using the calibration
of the record.
3. Calculate the average and maximum frequency.
4. Measure the duration of multipotential activity using the calibration of the record.

Calculations:

Conclusion:

57
 EMG of the m. biceps brachii

Task:
1. Measure the EMG activity of a muscle during its contraction with and without a load
using surface electrodes.
2. Evaluate the duration and intensity of the measured EMG and explain the differences.

Procedure:
1. Place the electrodes on the arm according to Fig. 4.16 (try to keep a
distance of 3 to 5 cm between the individual electrodes). Connect the
connectors according to particular colors.
2. After starting the EMG record, repeat the flexion (5-times) of the
hand without a load.
3. Repeat the measurement by flexing the hand with a load.

Fig. 4.16: Proper EMG

electrodes placement.

stick EMG record here

Conclusion:

58
 Determination of rheobase and chronaxie

Task:
1. Determine the threshold intensity of the electric current (i.e., rheobase) that caused the
response in the form of the muscle contraction (abductor digiti minimi – little finger
abductor) under different conditions.
2. Determine a duration (i.e., chronaxie), which is needed to elicit a response to a stimulus
with an intensity of 2-times of rheobase.
3. Write the values in the table and decide which measurement corresponds to a healthy
muscle, which one to the injured person with reduced irritability, and which one to the
situation after rehabilitation (recovery).

21
20
19
18
17
Stimulation intensity [mA]

16
15
14
13
12 Abductor
11 digiti
10 minimi
9
8
7
6
5
4
3 muscle A
2 muscle B
1
0 muscle C
3 20 30
0,01 0,1 0,3 0,5 1 10 100
0,05
Stimulation time [ms]

Table of recorded values:

Rheobase Chronaxie
value unit value unit
muscle A:
muscle B:
muscle C:

Conclusion:

59
 Measurement of the response of the median nerve

Task:
1. Use electromyography (EMG) to visualize sensory and motor responses in the reflex
arc of n. medianus using surface electric stimulation.
2. Determine the minimum value of electric current in mA that will elicit a sensory and
motor response, the value of latency (ms), amplitude (mV), and duration of the
response (ms).

Procedure:
1. Attach the recording electrodes to the examined palm: active (black) – thenar
eminence, reference (red) - thumb joint, earth (hook-and-loop touch fastener) -
distal part of the wrist (Fig. 4.17).
2. Place the stimulation electrode between the tendons approx. 8 cm from the active
electrode (black electrode towards the recording electrodes) and gradually apply el.
stimuli of varying intensity with 0.1 ms pulse duration. Stimulate up to maximum
sensory and motor response.
3. Write the obtained values in the table and compare the sensory and motor response
(Fig. 4.18).

Fig. 4.17: Electrodes placement. Fig. 4.18: A) Motor response (muscle action potential),
B) Sensory response (neuronal action potential).

Table of recorded values: Motor response Sensory response

value unit value unit
Min. value of el. stimulus
Amplitude
Latency
Response duration

Conclusion:

60
 5 5 Basics of thermodynamics

Thermodynamic laws
Thermodynamics is a discipline of physics, describing the laws of heat and thermal
processes. It involves relationships between quantities characterizing the macroscopic state
of the thermal system and changes in these quantities in physical processes associated with
heat exchange between the system and its surroundings. Thermodynamics deals with the
laws of conservation and conversion of energy applicable to thermal processes.

Thermodynamics is based on three main laws of thermodynamics:

1st law of thermodynamics (energy conservation)
The increase of the internal energy of the system (ΔU) is equal to the sum of the work (W)
performed by the surrounding bodies acting on the system and the heat (Q) transferred by
the surrounding bodies to the system.
∆𝑈 = 𝑄 + 𝑊

If the system receives energy: W > 0 and/or Q > 0; ΔU > 0

If the system transmits energy: W <0 and/or Q <0; ΔU <0
Another formulation of the 1st law of thermodynamics: It is not possible to construct a
"perpetuum mobile" of the first kind (i.e. a machine that cyclically repeats a process with
only initial energy).

2nd law of thermodynamics

It is not possible for heat to spread spontaneously from a colder place to a warmer one.
Another formulation of the 2nd law of thermodynamics: It is not possible to construct a
perpetuum mobile of the second kind (i.e. a cyclically operating heat engine that would only
receive heat from a warmer body and perform as much work as the received heat). This
means that heat energy cannot be spontaneously (and completely) transformed into mechanic
work.

3rd law of thermodynamics

The entropy change of the system equals zero in any isothermal process taking place at an
absolute zero temperature (in other words, it is not possible to reach an absolute zero
temperature).

Thermodynamic system - a body or group of bodies whose condition we are

investigating. Quantities by which the state of the system is determined, e.g. pressure,
temperature, volume, energy, are state variables.

61
 Thermal energy – is a part of the internal energy of the body (or defined part of space),
which is related to the disordered (thermal) movement of particles in the substance (in
defined space).

Temperature – a thermodynamic (state) quantity that characterizes the ability of an

object to receive heat spontaneously from another object. Temperature as a state quantity
describes one of the thermodynamic parameters - thermal equilibrium or imbalance. The
temperature units are °C or K or other. The following applies to the conversion between the
Celsius and Kelvin scales:
𝑇[𝐾] = 𝑡[°𝐶] + 273.15

Heat – is an energy that is transferred in a certain form, can be exchanged between

objects, or transformed from one to another form of energy or work. Heat (Q) is a change in
the thermal energy of an object. The unit of heat (as well as energy) is joule (J).
∆𝑄 = 𝑚. 𝑐. ∆𝑡

where: ΔQ is the heat change; Δt (K) temperature increase; m (kg) is the weight; c (J
/ kg.K) is the specific heat (material constant).

Thermodynamics of the living systems

The biological thermodynamic system has a complex structure and high order.
The order of the biological system means: 1. the order of the organism made of cells, 2. the
order of biopolymers, 3. the order of the structure of proteins and nucleic acids. Calculations
show little effect of biological order on entropy changes.
Entropy is a physical quantity that measures the disorder (randomness,
"clutter", degree of uncertainty) of a system. It is one of the state quantities in
thermodynamics, but it is introduced (more generally) in statistical physics. Its unit is J / K.
The second thermodynamic theorem states that the entropy of an isolated system
increases with time. For example, when we put a cold teaspoon in a cup of hot tea, the
entropy is less than after a certain time when the temperatures equalize. This can be
explained by the fact that the internal energy initially concentrated in the hot tea was later
distributed more evenly (and thus more disordered) between the tea and the teaspoon. And
the bigger the disorder, the bigger the entropy.
The entropy of a human organism, consisting of approximately 1013 cells, is practically
the same as the entropy of a set consisting of 1013 single-celled organisms. If we consider
the internal arrangement of the cells, which contains about 108 components of proteins,
nucleic acids, phospholipids, etc., then the arrangement of the human body corresponds to a
reduction in entropy of approximately ΔS ~ 1300 J / K. Such a decrease in entropy can be
compensated by simple physical or chemical processes, e.g. an increase in entropy of
approximately 1300 J / K occurs when 170 cm3 of water evaporates or 900 g of glucose is
oxidized.

62
 The living matter has low entropy because it is an open system that can receive
substance or energy from the environment. If a living organism receives substances with a
sufficiently high free energy from the environment (or a quantum of light during
photosynthesis in plants), some of them will use it to preserve it and some to increase the
organization and elimination of metabolic products. The low entropy states can be
maintained indefinitely if the system can reach steady states and thus the negative entropy
flow from the environment can be maintained in a high degree of state at the expense of the
environmental order.
An important feature of living systems is the creation of nonequilibrium steady states.
Assuming a local equilibrium, the biological system is developed to a steady state over time
so that its entropy production decreases over time until it reaches a minimum in the steady
state.
Thermodynamics is of great importance for the calculations of thermal coloration - the
synthesis of enthalpy of chemical reactions that take place in the body. Enthalpy is a
quantity that expresses the energy content of substances involved in a reaction that
takes place under unchanged pressure. For example, it allows you to determine how much
heat is released when nutrients are completely metabolized to CO2 and H2O. Although a
number of complicated reactions take place, their resulting thermal coloration can be
determined based on Hess's law, which defines that the thermal coloration of a story does
not depend on its path, i.e. on the intermediate stages, but on the final and initial state of the
system.
As well as the amount of heat, it is possible to determine what the maximum amount
of energy in a given chemical reaction can be converted into useful work, by calculating the
free enthalpy. According to the law of action (Guldberg - Waage law), a certain chemical
reaction A + B ↔ C + D takes place in both directions and is characterized by the equilibrium
constant K. At dynamic equilibrium, K = ([C].[D]) / ([A ].[B]), where [A], [B], [C], [D] are
molar concentrations in mol.l-1. The development of the reaction is influenced by the
environment (type of solvent, pressure, temperature) and the energy balance at constant
pressure and temperature described by free enthalpy, for which the change is ΔG = ΔG0 +
R. T. ln K. The change in ΔG0 corresponds to the standard state, where the components have
a unit concentration, a temperature of 298 K, and a pressure of 100.3 kPa. From the value of
ΔG it can be told whether the given reaction can take place in the given direction
spontaneously (ΔG < 0), or whether it is necessary to supply energy from another reaction
(ΔG > 0) in the given direction. For many reactions taking place in living matter, ΔG > 0,
but they are always part of cycles whose total change in free enthalpy is ΔG < 0.
As a result of the supply of nutrients, events are constantly taking place in the living
matter, which act against the thermodynamic balance and thus keep the organism alive.
However, no process, including developmental leaps during the evolution of the organism,
contradicts the basic thermodynamic postulates. Only after death, a complete state of
equilibrium occurs, because all thermodynamically possible events take place
spontaneously.

63
Transformation and accumulation of energy in living systems

The organism as an open system is in constant interaction with its surroundings.

Substances, energy, and information are being exchanged. Under normal conditions, the
body obtains a substantial part of its energy from food in the form of chemical energy. A
smaller part of the energy from the environment consists of chemical, light, mechanic and
thermal energy, causing irritation of the relevant receptors and consequently adequate
perception. In this case, the decisive factor is the amount of information transferred that will
affect the behavior of the system, but not its energy balance.
The energy delivered to the body is either accumulated for later use in the form of
chemical energy or is used directly to maintain all life processes in the body, i.e. it changes
into muscle work, chemical or electric energy, and heat. Energy is accumulated in the long
term by the storage of adipose tissue or in the short term by the resynthesize of macroergic
phosphates (ATP from ADP). Heat in the body arises mainly as a side product of the
conversion of other forms of energy. Only exceptionally it is generated in a targeted manner,
e.g. in the case of cold shake as a mechanism of thermoregulation. In order that this mostly
“waste” heat not to accumulate in the organism and thus not increase the temperature, the
organism is able to regulate its removal to the environment from a certain extent
(thermoregulation).
The primary source of energy for all living organisms living on Earth is either light (in
plants and photosynthetic bacteria) or energy released during chemical reactions during
nutrient processing. From the total energy of solar radiation (0.4 - 1). 1022 kJ per year is
about 0.5 % consumed in photosynthesis in the biosphere and the rest is back-radiated into
space. Chemical processes in cells take place through a large number of intermediate steps
catalyzed by specific enzymes. Electron transfer processes mainly provide energy for
individual processes. Macroergic phosphates play an important role in the transformation of
energy in living matter, the most important of which is ATP (adenosine triphosphate). It is
formed by various types of phosphorylation in the living system using the chemical energy
of nutrients or quanta of solar radiation. It can be produced by substrate phosphorylation
(anaerobic oxidation of sugars), oxidative phosphorylation (in mitochondria), or
photosynthetic phosphorylation (in plant chloroplasts). It is the universal storage of energy
in plant and animal cells, and in the opposite process, enzymatic cleavage of ATP, energy is
released again.
Response:
(𝐀𝐓𝐏)𝟒− + 𝐇𝟐𝐎 → (𝐀𝐃𝐏)𝟑− + (𝐇𝐏𝐎𝟒)𝟐− + 𝐇+

corresponds (at pH = 8) to a change in free enthalpy ΔG0 = -31 kJ / mol and the free enthalpy
obtained in this form is used to carry out the individual coupled endothermic reactions.
There are other energy-rich phosphate compounds. Upon hydrolysis
phosphoenolpyruvate is ΔG0 = -61.8 kJ / mol or sn-glycerol-3-phosphate ΔG0 = -9.2 kJ /
mol. The ATP - ADP system is practically in the middle of the scale and therefore acts as an
ideal donor-acceptor pair in the transfer of phosphate groups. An interesting fact is that the
daily consumption of ATP in an adult is about 70 kg on average. Basically, the human body

64
turns over its weight so it is clear that ATP must be synthesized almost constantly. For
example, in the case of the bacterium Escherichia coli, its supply is sufficient for only 2
seconds. ATP is applied in a manner similar to ensuring the course of endothermic chemical
reactions even during mechanic muscle work or active transport of substances.
Calorimetry deals with measuring the energy requirements of an organism and
determining the energy value of foods that meet these requirements. Heat is released by
burning food energy in the presence of oxygen. The heat of combustion of different nutrients
is different, its magnitude for proteins and sugars is about 17 MJ / kg, and for fats about 38
MJ / kg. As all the energy released in the body is eventually converted into thermal energy,
the amount of energy turnover can be determined by direct or indirect calorimetry. In
direct calorimetry, a living object is placed in the isolated space of a calorimeter and the
generated heat is measured by the temperature of a specified amount of water circulating in
its walls. In indirect calorimetry, the proband's oxygen consumption during respiration as
well as CO2 expenditure is determined, the energy equivalent is determined from their ratio,
and the magnitude of basal metabolism is determined from the tables.
The total energy turnover in an adult at an average daily activity is approximately 11
MJ / day. It is assumed that the value of total turnover in humans and higher animal species
is not proportional to the total body weight, but its surface area, so that per 1 m2 surface area
turnover is about 5 MJ / day. Individual organs are involved in metabolism variably (e.g.,
the kidneys show about 25 times more energy per unit weight than the whole body on
average). The intake of heat from the environment, which would lead to an increase in the
energy content of the body is very small. In cold-blooded (poikilothermic) animals, changes
in ambient temperature affect the reaction rate of many chemical reactions taking place in
the body. A substantial part of the energy is supplied to the animal in the form of chemical
energy, in plants that are capable of assimilation, light energy contributes a significant share.
In addition to this substantial part of the energy from food, which covers the energy
consumption of the body, a certain small amount of energy comes into the body through the
reflex arc in various irritations, which we register as a perception. In different irritations,
different types of energy (chemical, mechanic, thermal, electric, light) are converted into
electrical energy.
The speed of the chemical reactions that take place in the body is affected by body
temperature. In warm-blooded (homoiothermic) animals, the body temperature (internal) is
kept at a certain level - in humans around 37 °C. The condition for maintaining a constant
temperature is that the amount of heat generated in the body by metabolizing nutrients and
muscle work is equal to the amount of heat released into the environment. This balance is
maintained mainly by regulating the rate of heat release to the surroundings and little by
regulating the amount of produced heat. Heat is released from the body by the skin and lungs.
Inside the body, heat exchange is maintained mainly by blood flow. Arterioles in particular
play an important role. These vessels with smooth muscles in their walls can actively
contract (narrowing of the lumen - vasoconstriction) or relax (widening of the lumen -
vasodilation). The content of subcutaneous and visceral fat significantly affects the processes
of heat intake and transfer. The thermal conductivity of other tissues does not have a
significant effect on heat exchange. Four processes contribute to the heat loss of the organism

65
in varying degrees: radiation (in the form of infrared photons), convection (flow),
conduction of heat, and evaporation of water.

Measurement of the organism's energy expenditure

The human body has a stable internal temperature. In order for such an organism to
survive in nature with very variable temperatures, it is equipped with effective mechanisms
to maintain the internal temperature. Collectively, we call them thermoregulation. Heat
distribution in the body is accomplished by the changes in the blood flow. Changes in the
flow of the blood through the periphery, especially the blood supply to the surface layer of
the skin, change the surface temperature of the body and thus also the heat expenditure. Heat
exchange significantly depends on the difference between ambient and surface temperatures.
Therefore, in the cold, the skin is cold too (especially the peripheral parts such as the limbs
to reduce heat loss). Conversely, in a warm environment or with an excess of heat in the
organism (exertion, fever) the temperature of the periphery rises. If the heat expenditure is
insufficient, the body excretes sweat, which is evaporated. It consumes a considerable
amount of heat. Heat expenditure also depends significantly on the air flow and humidity.
Humans also respond to the temperature conditions with their conscious activity -
they wear clothes, hide from the heat and cold, etc. The thermoreceptors provide information
about the ambient temperature that mediate the feeling of cold and warmth (different types
of receptors detect warmth and cold). However, information about the warmth or cold feeling
is not objective. It is influenced not only by the temperature of the organism and the
environment but also by the condition of the organism itself, whether it was previously in a
warm or cold environment, air flow, air and skin humidity, etc.
The cooling power is a certain objectification of heat expenditure. It is a
meteorological parameter independent of the thermoregulatory and other reactions of the
organism. It only includes the external physical conditions of the heat exchange. The cooling
power is the amount of heat loss from the area unit (1 m2) of a physically defined body
with a temperature of 36.5 ° C per unit time (1 s) when it is exposed to the atmosphere.
It means that it is affected by air with a certain temperature and humidity, air flow, thermal
radiation, etc. The unit of cooling power is J. m-2. s-1 (as for each energy flow).
The measurement of cooling power is carried out by a katathermometer. It is an
alcohol-containing thermometer with a large measuring container and a reservoir in the
upper part (Fig. 5.1). The time required for the temperature to drop from 38 °C to 35 °C is
measured. These temperatures are directly marked on the katathermometer. Before the
measurement, the katathermometer has to be heated so that the measuring substance partially
rises into the upper reservoir (the column of the substance in the capillary must not be
interrupted).
Cooling power CP is determined from the measured time (t) and the constant of the
katathermometer (K).
K
𝐶𝑃 = [J. m−2 . s −1 ]
𝑡

66
 The cooling power depends mainly on the temperature of the air to which the heat is
transferred and the speed of airflow. Using the cooling power, we can calculate a person's
daily loss of heat (DLH). It states how many Joules of heat we lose without increased
physical activity under the same conditions as the measurement of the cooling quantity (if
we do not prevent it and the body does not reduce it by regulatory mechanisms).
DLH is calculated:
DLH = 𝐶𝑃. 𝑆𝑏 . 86400 [J]

Sb [m2] - the surface area of the body (find the table in the Annex chapter)
86400 [s] - number of seconds per day

38 °C

35 °C

Fig 5.1: Katathermometer.

67
 Measurement of cooling power, calculation of DLH

Task:
1. Using a katathermometer measure the cooling power in the room in quiet air and
during ventilation.

Procedure:
1. Prepare the katathermometer for measurement by heating it in hot water.
2. Measure the temperature drop time from 38 °C to 35 °C under quiet conditions.
3. Prepare the katathermometer for a new measurement.
4. Measure the temperature drop time from 38 °C to 35 °C during ventilation.
5. Calculate cooling power for both conditions and calculate the daily loss of heat.

Table of recorded values:

value unit
Time (quiet air):
Time (ventilation):

Calculations:

Conclusion:

68
The exchange of the heat, thermoregulation

Body temperature is given by the balance between heat generation by the body, heat
intake from the external environment, and heat expenditure from the body -
thermoregulation. Humans belong to warm-blooded (homoiothermic) animals, whose
internal temperature is maintained in a wide range of ambient temperatures at a stable level.
A stable temperature is maintained only in a certain central part of the body called the
thermal core of the body (the organs of the thoracic and abdominal cavities). Tissues on the
periphery around the core, typically limbs and skin, are called the thermal shell. The
temperature of the shell is unstable and usually lower than that of a core. The difference in
temperature in different parts of the shell is mainly due to different blood flow. The limbs,
especially the toes, show the largest fluctuations in blood flow, and thus a large fluctuation
in their temperature (Fig. 5.2).

Fig. 5.2: Body temperatures at different ambient temperatures.

The range of thermoregulation is determined by the lower and upper critical ambient
temperature at which the body can still maintain thermal balance. The skin, which acts as a
thermal insulator (fat layer), has good thermoregulatory properties. As a result of
vasodilation (dilation of blood vessels), the blood flow through the periphery increases, and
the blood flows closer to the body surface heating it and thus releasing more heat. In
vasoconstriction (narrowing of the blood vessels) the blood flow through the periphery is
restricted, the blood flows deeper from the surface and the heat expenditure decreases.
Thermoregulation is also carried out by changing behavior (choosing a suitable environment

69
and activity, taking the optimal position of the body), changes in metabolism, and muscle
activity (muscle tremor). If the air temperature exceeds the skin temperature, the body
receives heat from the air (e.g. solar radiation, radiation from warmed objects). In our
conditions, the air temperature is usually lower, and therefore the body does not receive heat,
but on the contrary, heat is released from the body. There are four physical mechanisms:
conduction, convection, radiation, and evaporation (Fig. 5.3).

Conduction

Conduction is the exchange of heat when bodies with different temperatures

come into contact. It is the transfer of the kinetic energy of the molecules of one object to
the molecules of another object (or in the object itself) by colliding with each other. The
thermal conductivity of different substances is directly proportional to their electric
conductivity. Metals conduct heat well, non-metals (e.g. textiles) are used as thermal
insulators. Heat exchange (expenditure from the body) by conduction is small in humans
and does not exceed 1% of the total heat expenditure. This is due to the low thermal
conductivity of the air and the fact that most of the objects with which we come into contact
conduct heat poorly (wood, textiles). Conduction occurs significantly in water, where the
heat exchange is about 23 times greater than in the air.

Convection

Convection (flow) is the exchange of heat in liquids and gases. It is caused by the
movement of particles with higher internal energy. In the body, heat is dissipated (and
distributed) by the flow of blood. It is distributed from organs such as the liver or muscles
to other parts of the body. An important mechanism is the dissipation of heat to the skin
capillaries (and from there to the surrounding environment, especially by radiation). By
convection, a person releases about 15% of the total heat exchange.

Radiation

Each body emits thermal radiation to the environment as infrared light. The
effective radiation area of the skin is about 80% of the actual surface in a sitting or standing
position and in a huddled position about 50%. The amount of radiated heat depends on the
temperature difference between the skin (the surface) and the surrounding objects (therefore
you can feel the coldness in a warm room with cold walls). In the case of a person at rest,
the loss by radiation in our conditions makes up 55 – 60 % of the generated heat.

Evaporation

Evaporation is a very effective mechanism of heat exchange. It is the only mechanism

that remains functional even when the ambient temperature is higher than the skin
temperature (the heat is absorbed by other mechanisms). The most important evaporation

70
mechanism is sweating. Sweat excretion can reach up to 1.7 l per hour. The maximum daily
amount is about 12 liters. Evaporation is highly dependent on the humidity of the
surrounding air, while in a humid environment it decreases significantly. For example,
in a sauna, a person can withstand an air temperature up to 120 ° C (at a relative humidity of
3-5%) without an increase in body temperature, while a short stay in a humid room leads to
an increase in body temperature (in a steam bath at 50 ° C in 15 minutes by 1 ° C). Heat
capacity depends on volume, heat loss depends on the surface. For this reason, e.g., in
toddlers (relatively large surface area), the regulation of body temperature is more difficult
and the temperature range of their thermoregulation is significantly narrower. In extreme
conditions (e.g. in the desert), an active person may lose 8 - 12 liters of water per day by
sweating, 0.5 - 1 liters by urination, and 0.75 liters by respiration. Unless drinking is
completely replenished, diuresis decreases, and blood density increases.

Fig. 5.3: Mechanisms of heat exchange.

71
Hypothermia, hyperthermia

In warm-blooded animals, internal body temperature is maintained within the

appropriate range of biological homeostasis by thermoregulation. Thermoregulatory
mechanisms are activated when the dynamic balance between heat production and
expenditure is disturbed. The human normal body temperature is 36.8 °C (98.6 °F) and
is physiologically highest in the evening and lowest in the morning.

Hypothermia

Hypothermia is a condition in which the internal body's temperature falls below the
value needed for normal metabolism and bodily functions. Hypothermia can be divided into
three stages of severity:

Stage 1
Body temperature 1 - 2 °C below normal temperature (35 - 36 °C). Mild to severe
tremors (muscle tremors) appear. The person is not able to perform complex tasks with his
hands (so-called numbed hands). Vasoconstriction occurs, which reduces heat loss.
Breathing becomes fast and shallow. “Goose bumps” are formed, and the hair (smooth)
muscles are tensed in an effort to create an insulating air layer around the body (which is
limited in humans due to lack of hair, but useful in some species of animals). A person may
feel sick to their stomach and be very tired. A hypothermic person may perceive an apparent
feeling of warmth as if it was a remedy, but in reality, it is the beginning of stage 2. Another
test to determine if a person is entering stage 2 is to try to touch the little finger with their
thumb; if that doesn't work, it's the first degree of muscle dysfunction. Vision problems can
also occur.

Stage 2
Body temperature 2 - 4 °C below normal temperature (33 - 35 °C). The shaking
becomes stronger, poor muscle coordination is more pronounced. The movements are slow
and strenuous, accompanied by slight confusion, although the person may appear vigilant.
The superficial blood vessels further contract so that the body concentrates its remaining
resources to maintain the temperature of vital organs. The person becomes pale, and lips,
ears, fingers, and toes can turn blue.

Stage 3
Body temperature drops below 33 °C. Speech problems, unfocused thinking and
memory loss, inability to use hands, and stumbling are beginning to occur. Cellular
metabolic processes stop. Below 30 °C, the skin exposed to the cold becomes blue and
swollen, muscle coordination disappears, walking becomes almost impossible and the
person shows an unreasonable attempt to hide in a small indoor area or even stiffens (stops
responding). Pulse and respiration are significantly reduced, and ventricular tachycardia or
ventricular fibrillation may occur. The main organs collapse.

72
 First aid:
• place the person in a warm room, if possible,
• give him dry clothes or wrap him in warm blankets,
• serve warm and sweet beverages, not alcohol!,
• if the person has no other injuries, he can be given a lukewarm bath, where the
water temperature gradually rises.
Warming up must be gradual to avoid cardio-respiratory shock. It is not recommended
to drink alcohol before staying in the cold, as it acts as a vasodilator and increases heat loss.

Hyperthermia
Hyperthermia is a process in which the body is unable to get rid of excess heat,
usually due to a failure of thermoregulation. Overheating occurs when the body generates
or receives more heat than it is able to transfer to the environment. In the initial phase,
thermoregulation mechanisms move blood from the central parts of the body to the skin
(splanchnic and renal vasoconstriction with cutaneous vasodilation). Over time,
thermoregulation mechanisms fail. The heat stops being removed from the center, the
temperature rises, and heatstroke or even thermal shock is developed.
Hyperthermia in humans is manifested by an increase in cardiac output, peripheral
vasodilation, and sweating. During intense sweating, there is a large loss of water and
electrolytes, followed by dehydration, and a decrease in blood pressure, which can develop
until collapse (heat shock). Hypovolemia causes the insufficient blood supply to the skin,
which prevents heat transfer through vasodilation and sweating. Due to significant water
loss, hypertonic dehydration occurs with consequent renal failure.

Symptoms and signs of hyperthermia:

• red hot skin (dilatation of blood vessels, active hyperemia) dry or damp, depending on
the degree of hyperthermia, sweating is present, termination of sweating is
characteristic for heat stroke as a late symptom of hyperthermia;
• nausea, vomiting, headache, weakness (due to dehydration and direct effect of high
body temperature);
• orthostatic changes in blood pressure (dehydration and drop in blood pressure),
weakness and dizziness in a sudden standing up from a horizontal position;
• tachycardia and tachypnoea with respiratory alkalosis, in severe dehydration the blood
pressure decreases, which reflexively leads to vasoconstriction and the skin color
changes from red to pale;
• functional changes in the CNS: convulsions (including opisthotonus), changes in
mental status, confusion, hallucinations, balance disorders, delirium, coma;
• increased cerebral vascular filling (hyperemia), cerebral edema, increase in
intracranial pressure with consequent compression of cerebral vessels, and decreased
flow - CNS dysfunction;
• tissue damage occurs at temperatures above 42 °C, protein damage occurs
(denaturation), and increased membrane permeability;
• urinary system: hematuria, oliguria to anuria as symptoms of acute renal failure.

73
 At high ambient temperatures above 41 °C (forgotten child in the car), the direct effect
of high temperature and secondarily the disruption of fluid and electrolyte homeostasis leads
to irreversible brain damage, so in this case, it is necessary to help quickly - start cooling the
affected person rapidly!

Temperature measurement

In medical practice two basic types of medical thermometers are used – fast
measuring thermometer and maximal thermometer (Fig. 5.4). Both of them are mercury
thermometers calibrated from approximately 35 °C to 42 °C with a measurement precision
of 0.1 °C. The fast measuring thermometer takes instant body temperature. Beware: one
must read out the body temperature while the thermometer is placed within the place of the
measurement.
In contrast, the maximal medical thermometer has a narrowed place on its measuring
capillary. Increasing temperature pushes the mercury through this constricted place, however
when the temperature starts to drop the mercury column disrupts in the constricted place (as
a result of surface tension) and the thermometer shows the maximal measured temperature,
for a long time. Nowadays, body temperature is often measured by digital electric
thermometers.

Fig. 5.4: Maximum (left) and fast measuring (right) medical thermometer.

Types of thermometers:
• Liquid thermometer – uses the volume expansion of the thermometer liquid
(mercury, alcohol, etc.)
• Bimetallic thermometer – uses a bimetallic strip composed of two metals with
different length expansions. As the temperature changes, the strip bends, and this
movement is transmitted to the thermometer scale.

74
 • Gas thermometer – to measure the temperature uses the dependence of the gas
pressure on the temperature at a constant volume of gas, or the dependence of gas
volume on the temperature at constant pressure.
• Resistance thermometer – when measuring the temperature, it uses the dependence
of the electric resistance of the conductor (resistance temperature sensor) or
semiconductor (thermistor) on the temperature.
• Thermoelectric thermometer (thermocouple) – for temperature measurement uses
the thermoelectric phenomenon (electrons, which are carriers of electric current, play
a significant role in heat conduction). By changing the temperature of two different
metals, thermoelectric voltage changes occur.
• Radiation thermometer – designed for measurement of high temperatures based on
the laws of thermal radiation (Planck's Radiation law, Wien's law, Stefanov-Boltzman
law). It measures radiation from bodies to the environment (infrared light sensors or
homing missiles work on the same principle).
• Infrared thermometer – designed for contactless temperature measurement. It uses
the same principle as a radiation thermometer but works in the infrared region of the
electromagnetic spectrum. It is often equipped with one or more guide lasers. When
measuring with this thermometer, it is necessary to know the emissivity of the
measured surface. It is also called a pyrometer.

Even under physiological conditions, body temperature slightly varies and differs
according to the place of measurement. Human body temperature is normally measured
under the armpit (axilla, 36.3 °C), under the tongue (sublingual, 36.5 °C), or in the rectum
(rectally, 37 °C), or in the external auditory canal (tympanic temperature, about 37 °C). A
decrease in temperature below 36 °C is called hypothermia, if it is around 37.5 °C, it is
called subfebrility, and if it rises above 38 °C (e.g., in the case of the disease), then it is a
fever (febris).

Thermography

The principle of thermography is the registration of infrared radiation of thermal

changes occurring in the pathologically affected area of the human body. Thermography
records the energy of electromagnetic radiation emitted by the patient's body. This
radiation lies in the infrared region (wavelengths λ = 0.8 - 10 μm) and is not visible to the
human eye. The spectrum of infrared radiation depends on the temperature of the emitting
body and its surroundings. The examination is performed by an indirect (electronic) or direct
procedure. In indirect thermography, the radiation is detected by a special camera with a
centering system, detector, and equipment for pulse processing and image recording (Fig.
5.5). The principle of direct thermography is the conversion of invisible infrared radiation
into visible ones using liquid crystals. Cholesterol crystals are most commonly used in
medicine.

75
 Fig. 5.5: Infrared cameras.

Preparing the patient for the examination

The examined skin surface should be uncovered for at least 15 - 20 minutes before
the examination and freed from the tightening effect of the clothes (pressure causes
hyperemia). At the same time, the patient acclimatizes to the environment in which the
examination is performed, occupying the same position as in thermography. The
acclimatization time can be shortened by cleaning the examination area with alcohol. The
skim area of the measurement should not be palpated to avoid pressure hyperemia. Powder,
oils and creams must be removed from the skin. The patient needs to be reassured mentally.
Skin changes such as warts, scars, hematomas and ulcerations must be recorded in the
protocol. A breast examination is recommended 10 days after menstruation.
The examination room must be sufficiently large (approximately 4 x 4 m), with a
constant temperature (19 - 21 °C), without daylight. There must be no heater near the subject.
During thermography, homologous organs of the body (e.g. both breasts, both limbs) are
compared.

Clinical indications for thermography

For the diagnosis of the pathological process, the temperature difference between the
pathologic and healthy tissue is observed. It is done by unequal heat production, the rate of
its expenditure, the size and activity of the possible pathological process, and the standard
temperature of the skin (body). Although thermography is not diagnostic specific, it can
provide valuable information about the degree and dynamics of the process (Fig. 5.6).
Temperature differences are displayed on a gray or color scale. Cold areas are darker, warm
areas are lighter, or otherwise colored. The temperature of both halves of the body is not
completely identical, the differences sometimes reach up to 1-2 °C. The warmest is the trunk
and face, the limbs are colder. All skin folds are also warmer, e.g. axillae, inguinal areas,
and areas below the breasts. Thermography is used as a diagnostic method for skin infections
and disease processes in the breast and limbs.

76
Fig. 5.6: Thermographic imaging of various parts of the body in a pathological process.

77
 Measurement of body temperature by maximal and digital
thermometer

Task:
1. Choose a suitable place for measurement of body temperature on your
body (e.g., axilla, the ear) (Fig. 5.7).
2. Measure your body temperature and compare it with reference values
(36.3 °C - 37 °C).
Fig. 5.7: Axillary
Table of recorded values: temperature measurement.

maximal thermometer digital thermometer

value unit value unit

Conclusion:

Measurement of surface temperature by thermocamera

Task:
1. Turn on the thermal imaging camera and calibrate it for measurement.
2. Choose suitable places to measure the temperature on your body.
3. Measure your surface temperature and write down the values.
Table of recorded values:
part of the body value unit

Conclusion:

Conclusion:

78
Air humidity

Air is a mixture of nitrogen (78 %), oxygen (21 %), noble gases, carbon dioxide (0.04
%), and (under normal circumstances) also water vapor that comes from seas, rivers, living
organisms, etc. Water vapor is a permanent component of the gaseous mixture of air. It has
a fundamental effect on various processes in the air, not only on the entire circulation of
water in nature but also on the balance of radiation, cleaning of air, etc. Water vapor can get
into the air only at a certain temperature until the air is saturated (saturation represents a
different amount of water at a different temperature). If there is not enough water vapor in
the air to saturate it, the air is kept unsaturated. From a thermodynamic point of view, we
call this state nonequilibrium. Condensation occurs with any excess (above saturation) of
water vapor in the air (Fig. 5.8).

evaporation equilibrium condensation

state
Fig. 5.8: Humidity changes; A) unsaturated air; B) equilibrium; C) excess of water vapor.

The amount of water vapors in the air is expressed as the pressure of water vapors
(in pressure units - Pa) or as the content of water vapor (expressed in g / m3). Saturation of
air by water vapors corresponds to the maximum amount of water in the gaseous state in a
given space at a given temperature. This also applies to atmospheric air. The amount of water
vapor in the air cannot be greater than the saturated value at a given temperature.

The main parameters by which we express humidity:

• the pressure of water vapors (e) – partial pressure of water vapors in the air [Pa]
• absolute humidity (a) – density / amount of water vapors per m3 of air
a = 0,623 e / R.T [g/m3]
where: e – partial pressure of water vapors; R – universal gas constant; T – absolute
temperature.

79
• relative humidity (r) – the ratio of actual (e) and maximum possible (E) (saturation)
amount of water vapor. r = e / E [%]
• dew point (τ) – the temperature at which the air would be saturated with present (actual)
water vapor content (at the current temperature it is not yet saturated)

Measurement of humidity

The humidity is measured by a hygrometer. The hygrometer can be:

• absolute or mass – air is sucked in through a tube filled with a hygroscopic substance
(a substance capable of absorbing and retaining moisture) until the entire known volume
is exhausted. By weighing the tube before and after suction, the weight of the absorbed
vapor is determined and the absolute humidity is calculated.
• the relative – relative humidity is measured by determining the dew point - the
temperature at which water vapors would become saturated. Its achievement is
manifested by the dewing of the surface of the body. This is how the Lambrecht
condensing hygrometer works (Fig. 5.9)
• psychrometer – relative humidity is measured using two thermometers, one is dry and
the other wet. Wet indicates a lower temperature (except in a situation of 100%
humidity). The higher the temperature difference, the lower the relative humidity (Fig.
5.10)
• hair hygrometer – it is a less accurate hygrometer based on a change in the length of
defatted hair, with a change in relative humidity (Fig. 5.11)

Fig. 5.9: Lambrecht hygrometer. Fig. 5.10: Psychrometer.

80
 The effectiveness of thermoregulation mechanisms depends on humidity. The
ideal relative humidity is considered to be 40 – 60 %, and long-term low humidity (below
40 %) can be harmful, especially in people prone to upper respiratory tract diseases
(inflammation) or in people suffering from reduced mucosal function (dry mucosa). The
effect of high air humidity (above 60 %) is tolerated by humans well and rather humid air is
acceptable for humans. However, the air with high humidity and ambient temperature over
22 °C is a breeding ground for the growth of fungi and bacteria and a risk factor for various
diseases, which is especially dangerous for asthmatics and people with reduced immunity.

Fig. 5.11: Hair hygrometer.

Measurement of air humidity, determination of dew point

Task:
1. Determine the partial pressure of water vapors in the air in the practical room by means
of a psychrometer.
2. Calculate other characteristics of air, absolute and relative humidity, and dew point.

Procedure:
1. Dampen the muslin on the wet (blue) thermometer.
2. Extend the fan spring and keep it running.
3. After the temperatures have stabilized, read the temperatures of the dry (red) and wet
(blue) thermometers. Write the measured values in the table.
4. Using a barometer determine the atmospheric pressure p.
5. Find the pressure of saturated water vapors for the “wet” thermometer Ew (see table in
the Annex).
6. Calculate the partial pressure of water vapors e.
7. Calculate absolute and relative humidity, and determine the dew point.

81
 The partial pressure of water vapors:

𝑒 = 𝐸𝑤 − K. 𝑝. (𝑇 − 𝑇𝑤 )

where: Ew – the pressure of saturated water vapors for “wet” thermometer, K – psychrometric
constant of the device (K=6,62.10-4 K-1), p – current atmospheric air pressure, T –
temperature of the dry thermometer, Tw – temperature of the wet thermometer

Absolute air humidity:

2,2. 𝑒 g
𝑎= [ 3]
𝑇 m
where: e – partial pressure of water vapors, T – temperature of dry thermometer in Kelvins

Relative humidity:
𝑒
𝑟= . 100 [%]
𝐸

where: E – the pressure of saturated water vapors for a “dry” thermometer

Table of recorded values:

quantity value unit
The temperature of the dry thermometer:
The temperature of the wet thermometer:
Atmospheric pressure:
The pressure of saturated water vapors for
“dry” thermometer:
The pressure of saturated water vapors for
“wet” thermometer:

Calculations:

Conclusion:

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 6 6 Respiration

Breathing (respiration) is one of the vital functions of living organisms. Breathing

is the process of exchanging oxygen (O2) and carbon dioxide (CO2), the main respiratory
gases, between the external environment and the cells. During respiration, due to the
contraction of the respiratory muscles, oxygen enters the pulmonary alveoli (their area is
about 70 m2 in a healthy adult) and is diffused into the blood. Hemoglobin within red blood
cells distributes the oxygen around the body to reach the tissues. It is used to oxidize nutrients
and release energy. The end product of these oxidative reactions is CO2, which travels in the
opposite direction to O2. It travels from the cells to the blood, to the pulmonary alveoli, and
subsequently is exhaled from the lungs into the surrounding air (tidal volume).

Breathing can be divided into:

• external – represents the process of exchange of O2 and CO2 between an external
environment and the blood,
• internal – represents the exchange of O2 and CO2 between the blood and the tissues
(cells),
• cellular respiration – respiration at the cellular level (energy production in
mitochondria).

The basic physical laws describing the mechanism of respiration are:

• Dalton's law – total pressure of a mixture of non-reactive gases (P) equals the sum of
the partial pressures of individual gases in the mixture p (P = p1 + p2 + p3 ...). It is
mainly used to calculate the concentrations of individual breathing gases, where the
higher the gas concentration (volume %) in the gas mixture, the higher its partial
pressure. A commonly used unit is kilopascal [kPa]. (Fig. 6.1)

Fig. 6.1: Non-reactive mixture of the gases representing the Dalton's law at the sea level altitude.

• Fick's law – the law of diffusion, which is primarily a mechanism of passive transport
of substances. Diffusion related to respiration depends on the gradient of breathing
gases O2 and CO2 partial pressures in the alveolar air and the pulmonary bloodstream
83
 (or difference in the blood and tissue for internal breathing, Fig. 6.2). In the lungs, at
the alveolar-capillary membrane, the pressure gradient of O2 equals to 8 kPa and for
CO2 approximately 0.8 kPa.

Blood plasma

Fig. 6.2: LEFT: Representation of external breathing. RIGHT: Representation of internal breathing.

The diffusion of O2 and CO2 across the alveolar-capillary membrane is driven

by Fick's law:
𝑃1 − 𝑃2
𝑽 = k .𝐴
ℎ
where: k - diffusion constant; A - diffusion area (approx. 70 m2); h - membrane thickness
(0.8 µm); P1 and P2 - partial pressures of the gases.

• Henry's law (Henry-Walton) – the amount of gas physically soluble in the liquid is
directly proportional to its partial pressure above the liquid and its solubility
coefficient (Fig. 6.3). There are about 3 ml of O2 physically dissolved in 1 l of arterial
blood and 197 ml chemically bond to hemoglobin in erythrocytes (there is in total 200
ml of O2 per 1 l of blood, i.e. 1000 ml of O2 in 5 l of blood in a body).
Low pressure

High pressure

Fig. 6.3: Partial pressure of gas above water level, equals to the partial pressure of the same gas in the
solution – the higher the pressure, the more gas is physically dissolved in the liquid.

84
Biophysics of breathing

The lungs are sac-shaped elastic organs located in the thoracic cavity. Alveoli
communicate with the outside environment through the airways and their total area (A) in
a healthy adult is about 70 m2. Breathing is based on a rhythmical rise and fall of the chest
volume. The lungs which do not contain muscles, copy these changes resulting in a change
in the lung volume. This process is called the respiratory cycle and has two phases:
inhalation or inspiration (active process) and exhalation or expiration (passive process at
rest). The membrane which covers a lung surface is named the visceral pleura and the other
one covers the inner side of the chest cavity as the parietal pleura (Fig. 6.4).

Fig. 6.4: Lung detail showing a visceral and a parietal pleura.

The space between two pleurae is filled by the pleural fluid to lubricate the membranes
(while they are sliding along each other during breathing). Pleural pressure is defined as
pressure within pleural space and under physiological conditions is negative compared to
atmospheric pressure. The negativity of pleural pressure keeps the lungs inflated and the
lungs follow the changes in chest volume during the respiratory cycle.
Inspiration is always an active process, caused by the activity of the respiratory
muscles, especially the diaphragm (main inspiratory muscle). Expiration is mostly a
passive process, caused by the elasticity of the lungs and chest tissues (chest and tissue
recoil forces). Only an extra effort makes the expiration active, with the engagement of
expiratory muscles activity (auxiliary muscles – neck, thoracic and abdominal muscles).
External respiration involves 4 basic processes:

85
 • ventilation – regular exchange of gases between the external environment and the
alveolar space, which is provided by respiratory muscles - diaphragm (works as a
piston) and the intercostal respiratory muscles (internal serve for expiration, external
for inspiration). Effortful expiration is also conditioned by muscle activity,
• intrapulmonary distribution – mixing of inspired fresh air with the air that has
remained in the airways after exhalation (anatomical dead space = 150 ml),
• diffusion – the movement of O2 and CO2 through the alveolocapillary membrane
(Fick's law) following their partial pressure gradients,
• perfusion – permanent blood circulation in the pulmonary system. O2 and CO2 are
physically dissolved in blood plasma and chemically bound to erythrocyte
hemoglobin.

The respiratory system consists of the lungs, airways, and respiratory “pump piston”
consisting of the chest wall and respiratory muscles. As mentioned above, the lung tissue
does not contain muscles, so the chest and respiratory muscles provide either underpressure
(inspiration) or overpressure (expiration). The main respiratory muscle is the diaphragm.
The diaphragm is a flat, upward-curved dome-shaped muscle separating the thoracic and
abdominal cavities. Its surface area is about 250 cm2 in a healthy adult and provides 60-75%
of the air exchange in the lungs. It acts as a pump piston during the breathing cycle. With a
calm breath, it decreases downwards, by about 1 - 1.5 cm. A 1 cm decrease of the diaphragm
will increase the chest cavity volume by approximately 250 ml. With a calm exhalation, the
diaphragm returns passively to its original position leading to a reduction in chest cavity
volume (due to the retraction/recoil force of the lungs and thorax). During strong inspiration,
the diaphragm can decrease by up to 7 cm. During a forceful expiration, it moves upwards
mainly by the contraction of the abdominal (auxiliary) muscles creating abdominal pressure.
The inspiratory respiratory muscles include the diaphragm and the external
intercostal muscles (musculi intercostales externi). During inspiration, the diaphragm
contracts, flattens, and moves downwards and enlarges the volume of the thoracic cavity).
The fibers of the external intercostal muscles run obliquely from top to bottom, starting at
the upper rib with a proximal attachment closer to the spine and ending with a distal
attachment further from the spine. During their contraction, the ribs and sternum rise
upwards and forwards. Lifting the upper six ribs widens the anteroposterior diameter of the
chest and lifting the lower six ribs widens the transverse diameter of the chest.
The expiratory muscles include the abdominal muscles and the internal intercostal
muscles (musculi intercostales interni). The muscle fibers of the internal intercostal muscles
run obliquely, but in the opposite way to the external respiratory muscles (Fig. 6.5). Their
distance on the proximal rib is further from the spine than the attachment on the distal rib.
These muscles rotate the ribs so that the sternum moves downward during the expiration and
the chest diameter decreases. During expiration with maximal effort (or during reflexes such
as coughing or sneezing etc.), the auxiliary abdominal muscles compress the abdomen. The
positive abdominal pressure pushes the diaphragm up into the thoracic cage to minimize the
chest volume.

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 Fig. 6.5: Intercostal muscles.

Parallelogram

The activity of the intercostal respiratory muscles during the respiratory cycle can be
demonstrated using a simple model - parallelogram. The parallelogram model (Fig. 6.6)
consists of a longer stick that represents the spine. Attached to it are two shorter slats, which
represent two neighboring ribs. These are connected by a shorter rod in front (as opposed to
a longer bar) representing the sternum. Between the upper and lower ribs, there are stretched
rubber bands that simulate the intercostal muscles. The rubber band attached closer to the
spine on the upper rib and closer to the sternum on the lower rib, represents the external
intercostal muscle (m. intercostalis externus), and the second rubber band, which is attached
to the upper stick near the sternum and the lower stick near the spine represents the internal
intercostal muscle (m. intercostalis internus).

Fig. 6.6: Model of parallelogram.

The intercostal muscles are represented by rubber belts. During muscle contraction,
when the length of the muscle is affected, the movement demonstration on the parallelogram
should be presented by shortening of the rubber belt (contraction of the muscles). When the
internal intercostal belt is shortened, the force causes downwards motion of both ribs and
sternal bone. The applied force is the same for the lower and upper rib; however, the force
87
momentum is different. The moment of the force affecting the lower rib at the place where
external intercostal muscles are attached is higher (the lower rib tries to get closer to the
upper rib). Because they are also attached to the sternum, it is not possible for the ribs to get
closer to each other. Thus, when external intercostal muscles contract, the ribs, and the
sternum move upwards (inspiration). When internal intercostal muscles contract, the ribs,
and the sternum move downwards (expiration).

Hering's model of breathing

Hering's model (Fig. 6.7) consists of a glass bell (1) (thorax), at the bottom of which
is a rubber membrane – diaphragm (2). The rubber diaphragm may be pulled downwards or
pushed upwards into the bell imitating the movements of the diaphragm. A glass pipe (3)
(trachea and upper airways) passes through the upper opening of the glass bell and further
splits into two pipes – bronchi (4). Elastic rubber balloons are attached to these tubes – lungs
(5). A water tonometer (6) demonstrates the pressure changes between the balloons and the
inner wall of the bell (pleural pressure). In the lower part of the glass bell, a fine rubber belt
is attached (7) imitating intrathoracic veins and walls of cardiac atria. The whole model is
situated on a metal stand enabling movements of the rubber membrane (diaphragm) during
“breathing”.
3

1
4

6 5 7

Fig. 6.7: Hering's model of breathing.

Experiments with Hering's model

Representation of quiet breathing and exhalation

When the rubber membrane moves downwards (Fig. 6.8 left) (movement of the
diaphragm during inspiration), the inner volume increases, with the depression of the pleural
pressure – a water in the water tonometer arm (red liquid, which is directly related to the
space of the bell) elevates on the bell side. At the same time, balloons and the thin-walled
rubber cylinder begin to inflate and increase their volumes. Similar changes occur in the
chest during inspiration. Due to the drop in pleural pressure in the pleural cavity, the same
decrease in pressure could be observed inside of the lungs (intrapulmonary pressure –
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negative intrapulmonary pressure makes the pressure gradient from the outside to the inside
of the lungs). Lungs are passively filled by the air and blood is sucked into the heart by
stretching the flexible walls of the large veins and the atria of the heart.
When the rubber membrane moves back upwards (Fig. 6.8 right) (passive movement
of the diaphragm during expiration), the inner volume of the glass bell decreases. The level
of the water in the tonometer (red liquid) reaches the same height (or inverts, see Fig. 6.8) –
a decrease in the volumes increases the pleural pressure. The volume of the balloons
decreases, and so does the volume of the rubber cylinder. Similar changes occur in the
thoracic cavity during expiration. The volume of the thoracic cavity decreases, the pressure
in the pleural cavity increases (it still remains negative during quiet breathing) and
intrapulmonary pressure reaches positive values (higher than atmospheric pressure) making
the pressure gradient from the inside of the lungs to the outer environment. As a result of
these changes, the air is exhaled from the lungs to the surroundings, and the blood flow in
the large thoracic veins decreases.

Fig. 6.8: Hering's model of breathing. LEFT: demonstration of calm inspiration. RIGHT: demonstration of
calm expiration (passive process).

Müller's experiment
Close the opening of the upper glass tube and at the same
time pull the rubber membrane (diaphragm) down firmly (Fig.
6.9). A large negative pressure is created inside the bell. The thin-
walled rubber cylinder (large veins in the chest) significantly
increases its volume. The intrathoracic negative pressure
considerably decreases when there is a maximal inspiration effort
with the vocal cords closed so that no air enters the lungs. Under
these conditions, the vacuum in the thoracic cavity can reach
negative values as low as - 5.3 kPa. The result is a massive suction
of blood into the thoracic veins and the right atrium of the heart.
Fig. 6.9: Müller's experiment.

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Valsalva's experiment
Close the opening of the upper glass tube and at the same
time push the rubber membrane upwards (Fig. 6.10). It creates a
massive overpressure inside the bell, which can be seen by the
water tonometer. The thin-walled rubber cylinder (large veins in
the chest) reduces its volume (due to the compression).
Intrathoracic pressure reaches high positive values of up to +6.7
kPa with maximum expiratory effort and closed vocal cords
(glottis). As a result of this overpressure, the vessels in the
thoracic cavity are compressed and the blood flow in the veins
and also the lungs is more difficult. Blood is then pushed into the
abdominal and jugular veins slowing the heart down. This
maneuver may be used as first aid for some types of Fig. 6.10: Valsalva's experiment.
tachycardia.

Lung volumes and capacities

Lung ventilation parameters provide important information in respirology

diagnostics. There are static (volumes and capacities) and dynamic (that change over time)
parameters.

The basic static lung volumes:

• tidal volume (VT) – the volume of the air inspired or expired during a quiet inspiration
and a quiet expiration. In a healthy adult, it reaches a value of 500 ml, which represents
12-18% of the vital capacity of the lungs.
• inspiratory reserve volume (IRV) – the amount of air that can be inspired (with
maximum effort) after quiet inspiration. It represents a volume of 2500 ml, which is
60% of the vital capacity of the lungs.
• expiratory reserve volume (ERV) – the amount of the air that can still be expired
(with maximum effort) after quiet expiration. It reaches a volume of 1000 ml, which
is 25% of the vital capacity of the lungs.
• residual volume (RV) – the volume of air that remains in the lungs after maximal
expiration (with maximum effort). RV cannot be measured spirometrically. It
comprises approximately 1 200 ml and consists of a collapse volume (400 ml, the
value of which the lung volume is reduced in pneumothorax) and a minimal volume
(800 ml, the amount of air that remains in the lungs after a collapse caused by a
thoracotomy).

Lung capacities are defined as a combination of several lung volumes. The static lung
capacities include (Fig. 6.11):

• vital capacity (VC) - the summation of tidal volume (VT), inspiratory (IRV), and
expiratory reserve volume (ERV). It is the amount of air that can be expired after

90
 previous maximal inspiratory effort. Its value depends on the volume of the thoracic
cavity, the expansibility of the chest and lungs, and the performance of the respiratory
muscles. There are also other parameters that affect VC: age, gender, height, and
weight of the person. The vital capacity of the lungs is a frequently used diagnostic
parameter in evaluating the performance of athletes in sports medicine or various
chronic obstructive (bronchial asthma) and restrictive (silicosis, anthracosis) diseases
of the respiratory system.
• inspiratory capacity (IC) – the summation of tidal volume (VT) and inspiratory
reserve volume (IRV).
• expiratory lung capacity (EC) – the summation of tidal volume (VT) and expiratory
reserve volume ERV,
• total lung capacity (TLC) – the summation of vital capacity (VC) and residual
volume (RV).
• functional lung residual capacity (FRC) – represents the amount of air that remains
in the lungs after quiet expiration (FRC = ERV + RV).

Fig. 6.11: Lung volumes and capacities.

Spirometry

Spirometry (Fig. 6.12) is the basic functional examination of the lung condition
known also as the breathing test. Spirometry, allows us to measure the static or the dynamic
ventilatory parameters, e.g., lung volumes, lung capacities, airflow, breathing rate, and
maximal voluntary ventilation. Spirometry gives us information on how much air can be
breathed out in one forced breath, as well as how easily and quickly this test can be
performed. In clinical practice, spirometry can be performed periodically to check whether

91
a treatment for a chronic lung condition was helping or for example in sports medicine to
check the training effects.

Indications for spirometric examination:

• diagnosis of lung disorders (breathing difficulties),
• diagnosis of specific lung diseases (bronchial asthma, chronic obstructive pulmonary
disease, and others),
• monitoring of lung function in workers exposed to harmful substances (asbestosis,
anthracosis),
• examinations before surgery, sports medicine, etc.

Rules for spirometric test:

• omit bronchodilators and sedatives before the examination (if the patient is taking the
given medicines),
• do not smoke, drink alcohol or caffeinated beverages 6 hours before the examination,
• wear loose (not tight) clothing for examination.

Procedure
The test lasts approximately 5-30 minutes. Prior to the exam, basic information such
as first name, surname, date of birth, gender, ethnicity, basic anamnestic data, and other
factors that can affect the spirometry (e.g., smoking, airway inflammation, etc.) should be
obtained. The patient puts a clip on his nose and a mouthpiece in his mouth, which is stuck
on a measuring turbine. A person has to take the mouthpiece in mouth tightly, so no air is
leaking away. Maximum inspiration is followed by a rapid and strong expiration. The
examined person takes an upright position and slightly leans forward during forced
expiration. The result of the test is the spirometric curve. The cooperation between the
examined person and the examiner is very important during the examination, otherwise, we
may get false positive/negative results.
For a vital capacity, the result determines the vital capacity measured (VCm)
usually given in liters. Then, we have to specify the so-called vital capacity standard (VCs)
reflecting the capacity that the person should have according to the anthropometric
parameters (mainly gender, age, height, and weight). The result is given in the form of a
percentage of VC obtained by the division of VCm/VCs. Percentages higher or close to
100% are normal. Percentages lower than 80% are considered abnormal.

Fig. 6.12: A person undergoing a spirometric test.

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 Demonstration of intercostal muscle function - parallelogram

Task:
1. Describe the parts of the parallelogram shown in the picture.
2. In the picture, use the arrows to mark the imitation of inspiration and expiration and
color-code the involvement of the muscles.

93
 Hering's model of breathing

Task:
1. Describe Hering's model of breathing and the effect of diaphragm movements on chest
pressure conditions.

Spirometry - measuring the vital capacity of the lungs

Task:
1. Measure the vital capacity of your lungs.

Procedure:
1. Put values of your height, weight, age and gender into PC.
2. Select the correct program to measure vital capacity. Take an upright position.
3. Put the mouthpiece in your mouth and close your nose with a clip.
4. Breathe calmly. After the audible signal, inhale as much as possible and exhale as
smoothly as possible.
5. Write your values in the protocol and compare them with the reference values in the
table.

Measured values:

Conclusion:

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 7 7 Biophysics of heart and circulation

Mechanic work of heart

The heart is a system of two pressure pumps (chambers) with two atria just before
them. The heart valves (mitral, tricuspid, pulmonary, and aortic valves) keep the appropriate
one-way direction of the blood flow, preventing it from flowing back. The heart works like
a pressure pump, which means that every time a heart muscle contracts, pressure builds up.
The contraction of the heart muscle is called systole (atrial, ventricular), its release
(relaxation) is known as diastole. The pressure in the atria is higher during diastole than in
the ventricles, which creates a pressure gradient enabling the blood to flow continuously
from the atria to the ventricles throughout the diastole of the ventricles. Systole and diastole
alternate regularly – heart cycle. During each cycle, the heart pushes out the blood volume
(ΔV) by pressure (p) performing a mechanic work:

𝑊 = 𝑝 Δ𝑉

The following applies to the kinetic energy (Ek) of systolic cardiac output:
1
𝐸𝑘 = 𝜌 𝑣 2 Δ𝑉
2

where: ρ – blood density; v – speed of blood.

Total mechanic work:

𝑊ℎ𝑒𝑎𝑟𝑡 = 𝑊 + 𝐸𝑘

Example: If the left ventricle displaces a blood volume of 70 ml at a pressure of 120

mmHg, then the work (p.V) of the left ventricle is: 15960 Pa. 70x10-6 m-3 = 1.117 J. The
work of the right ventricle at the same blood volume and pressure of 15 mmHg is 0.140 J.
At a blood density of approx. 1048 kg.m-3 and a blood flow rate of 0.5 m.s-1, the kinetic
energy of both chambers equals 0.018 J.
Then, the total work of the heart at rest is: 1.117 J + 0.140 J + 0.018 J = 1.275 J per cycle.

Cardiac cycle

The cardiac cycle represents the performance of the heart between two successive
beats. During the atrial diastole, the right atrium (atrium dextrum) receives deoxygenated
blood from the venous system (vena cava superior and inferior), while the left atrium
(atrium sinistrum) receives oxygenated blood from the pulmonary system (venae
pulmonales). Ventricular diastole and also atrial systole are associated with the blood flow
from right atrium to the right ventricle (through the tricuspid valve) and from the left atrium
95
to the left ventricle (through the mitral valve). These valves are also known as
atrioventricular or AV valves. Ventricular systole pushes the blood from the right ventricle
(ventriculum dextrum) to the pulmonary arteries (arteriae pulmonales) and from the left
ventricle (ventriculum sinistrum) to the aorta.

1 2 3 -4 5 6 7 1 2 3-4 5 Cardiac cycle phases

Fig. 7.1: Cardiac cycle figure demonstrates four main events: aortic pressure, left atrial pressure, left
ventricular pressure and left ventricular volume. Cardiac cycle phases: 1. Atrial contraction, 2. Isovolumic contraction,
3. Rapid ejection, 4. Slow ejection, 5. Isovolumic relaxation, 6. Rapid filling, 7. Reduced filling.

The cardiac cycle can be divided into 7 phases:

1. Atrial contraction: As atria contract, increased pressure inside of the atrial

chambers pushes the blood “actively” to the ventricles. However, about 75% of the
blood flows “passively” to the ventricles prior to the atrial systole (during the
diastole) by atrium-ventricle pressure gradient. Atrial contraction then forces 25% of
the resting blood to the ventricles (it depends on the actual demand, in the resting
conditions, the heart is filled passively for up to 90% and only 10% is provided by
atrial contraction). During the contraction, blood is not able to flow back to the
venous system because of the inertial effects of the venous return and also the
direction of the chamber contraction which points towards the AV valve and the
ventricle (also known as the “milking effect”). However, even without the blood
flow, the diastolic pressure in the venae cavae is higher compared to the atria. At the
ventricular volume curve (Fig. 7.1), atrial contraction appears as a notch starting just
above a P wave known also as “atrial kick”. At the end of this contraction, the valves
close (reversal pressure gradient). There is the end-diastolic volume in the ventricle
96
 at this time point, the volumes of both ventricles are maximized. Typically, the
volumes in resting conditions are 110-120 ml for each ventricle (depending on the
age and body surface).
2. Isovolumic contraction: In general, the isovolumic phase represents the time period,
where only the pressure increases while the volume remains the same. At the
beginning of the ventricular contraction, exceeded pressure closes AV valves to
prevent the blood from flowing backward (contraction of the papillary muscles with
the chordae tendineae, makes the first heart's sound). During this time, while the
aortic and pulmonary valves open, the intraventricular pressure increases without a
change in volume - no ejection. At the atrial pressure curve (Fig. 7.1), another notch
is visible during the isovolumic contraction. It is caused by the bulging of AV valves
back into the left atrium slightly increasing atrial pressure.
3. Rapid ejection: Aortic and pulmonary valves open. Accumulated energy forces the
blood to the aorta and pulmonary arteries to achieve maximal systolic pressures.
Initially, the atrial pressure decreases causing a negative pressure inside, while the
valves do not bulge upwards anymore. With the fall of the intra-atrial pressure, blood
starts to flow to the atria from their respective venous tracts. This causes the pressure
to increase which will continue to phase no. 5 where AV valves open, as can be seen
in the atrial pressure curve (Fig. 7.1).
4. Slow ejection: Ventricular pressure and ventricular volume decrease. Atria are filled
with blood.
5. Isovolumic relaxation: When the pressure in the chambers decreases, the reverse
pressure gradient closes the valves (the second sound of the heart). However, a small
amount of the blood escapes back to the ventricles, which slightly decreases the
blood pressure (this phenomenon is known as dicrotic notch as seen on the aortic
pressure curve). Relaxation of the muscle fibers results in the decrease of the
ventricular pressure without a change in the volume. The volume of the blood, which
remains in the ventricle after the systole is completed, is known as end-systolic
volume, typically 40-50 ml. The difference between end-systolic and end-diastolic
volumes is known as stroke volume representing the volume pumped to the system
(70–80 ml). The rest of the blood remains in the ventricles even after the systole was
finished. As the blood returns to the atria, the pressure in the atria increases.
6. Rapid filling: As the ventricular muscle fibers continue relaxation, the inner pressure
still decreases. When the ventricular pressure falls below the atrial pressure, the AV
valves open. The pressure gradient across AV valves leads the blood into the
ventricles. Thus, in phase 6, the pressure in the atria is higher than in the ventricles,
which creates a pressure drop and allows blood to flow “passively” from the atria to
the ventricles during most of the diastole. The rapid filling provides the sound
number three. Due to the continuous relaxation of the ventricular muscle fibers also
the intraventricular pressure continues to fall for the whole filling period. However,
the positive pressure remains in the pulmonary artery and the aorta even during the
diastole which is caused by the elasticity of the arteries!
7. Reduced filling: As the ventricles become filled, they are less compliant, which
leads to a rise in pressure. This decreases the pressure gradient across the AV valves,
reducing the blood flow and thus the speed of the filling. By the end of this phase,
just before phase 1, the ventricles are filled to about 70-90%. A new cardiac cycle
may begin.
97
Mechanics of blood flow

Blood circulation can be understood as a closed system that consists of the heart,
vessels (elastic arteries and veins) and blood. The heart is like an engine for the entire
circulation and pushes blood into the distributional (vascular) system. The circulation is
unidirectional, i.e., blood flows in one specific direction in each part. Pressure gradients
between the heart and blood vessels and between the arterial and venous blood systems are
necessary for continuous blood flow. The vascular system is flexible and capable of active
contraction (especially arterioles) – actively changing its lumen and affecting the blood flow.
Blood flow (Q [l/s]) depends directly on a pressure gradient and indirectly on
vascular resistance (especially arterioles) and blood viscosity. The relationship between
blood flow, pressure, viscosity and vascular resistance is expressed by Poiseuille - Hagen's
law:

Π . 𝑟 4 (𝑃1 − 𝑃2 )
𝑄=
8 .η .𝑙
where: Q - the amount of blood (volume), r - radius of the vessel, P1-P2 – pressure difference
(gradient), η - viscosity of the blood, l - length of the vessel.

From a clinical point of view, the Poiseuille - Hagen's law is important to calculate
the total resistance of the vascular bed of large and small blood circulation, based on the
measured values of cardiac output (Q) and the pressure gradient (P1-P2) in the vessels.
Of note: Increased vascular resistance causes an increase in systemic blood pressure
and thus increases the workload of the heart. The use of Poiseuille – Hagen’s law also
depends on the nature of the blood flow. Depending on the velocity, the viscosity of the blood
and the anatomical shape of the vessels, the blood flow may be either laminar or turbulent
(see Chapter 2 Properties of the liquids).

With each contraction of the heart, biochemical energy is converted into

hydromechanic energy, which is needed to maintain blood pressure. The heart performs its
work by pushing a certain volume of blood against the pressure in the aorta changing static
work to kinetic work. The total energy then depends on both the pressure and also the kinetic
force. The Bernoulli equation applies to the unit volume of fluid (blood) flow:

1
𝑝+ 𝜌 𝑣 2 = constant
2
where: p - fluid pressure, ρ - fluid density, v - flow rate.

The law of energy conservation applies. As the kinetic energy increases (flow in the
tube with a smaller diameter S2), the potential energy decreases – a drop in the fluid pressure
(h2). When blood flows through a smaller diameter vessel, the velocity is higher, but the
blood pressure is lower (and vice versa) - Bernoulli's law (Fig. 7.2).

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 Fig. 7.2: Bernoulli's law.

Mechanic properties of the vessels

During the heart systole, blood is pushed away from the left ventricle to the aorta and
the large arteries under a certain pressure, known as systolic blood pressure. Arteries are
elastic because elastic fibers in their walls and higher blood pressure are needed for them to
be stretched. The walls of the arteries are under tension and a part of kinetic energy is stored
within the elastic vascular walls thus converting it to potential energy. During diastole, this
potential energy is converted back into kinetic energy due to the movement of the elastic
vessel wall by elastic tension. The vessel elasticity is responsible for the diastolic blood
pressure whereas the blood continues to flow even during diastole (the heart is being filled
by blood). The transformations of kinetic to potential and back from potential to kinetic
energy occur continuously in the bloodstream. This phenomenon is known as the elastic
effect of the blood vessels. The stiffer (less elastic) the artery is, the higher the systolic
blood pressure during systole and the lower the diastolic flow during the diastole phase
of a heart (Fig. 7.3), e.g. in patients suffering from vessel atherosclerosis (decreased vessel
elasticity), both systolic and diastolic arterial blood pressures are above the normal values
and heart is overloaded.

Fig. 7.3: Comparison of elastic and non-elastic vessels.

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Pressures in the heart and surrounding vessels (approx. in systole/diastole):

Systolic/Diastolic Systolic/Diastolic
BP [kPa] BP [mmHg]
Right atrium + 0.6 / - 0.6 + 4.5 / - 4.5
Right ventricle 4.6 / 0 35 / 0
Pulmonary artery 4.6 / 1.2 35 / 9
Left atrium + 0.6 / - 0.6 + 4.5 / - 4.5
Left ventricle 16 / 0 120 / 0
Aorta 16 / 10.5 120 / 80

There is zero pressure in the ventricles of the heart during diastole – ventricles are
ready to be filled by the blood. In the pulmonary artery and the aorta, there is a positive
pressure in the diastole caused by the elasticity of the arteries (see the model of elasticity).

Arterial elasticity:
• maintains blood pressure in the vascular system during diastole - if the vessels were
not flexible, the blood pressure would drop to zero during diastole (in vitro),
• allows continuous blood flow in the vascular system during both systole and diastole,
• reduces the work of the heart while increasing its performance (efficiency).

Model of blood vessel elasticity

The vascular elasticity model (Fig. 7.4) consists of a jar with a colored fluid (blood
reservoir representing the left ventricle of the heart), where the pressure can be increased
using a balloon attached to it (pressurize the system). A tube representing the aorta, at the
beginning of which the clamp is located, emerges from the reservoir. The cardiac cycle
(systole and diastole) is simulated by pressing this clamp. Behind the clamp, the tube splits
into two branches, at the beginning of which there are one-way valves that allow fluid to
flow in one direction only. The first glass branch represents a nonelastic (rigid) vascular
system. The second rubber branch represents an elastic vascular system. Both are connected
to a small glass jar turned upside down. The liquid flows also through this container and
compresses the air inside of it – it is compressed by the kinetic energy of “systole” simulating
the potential energy store and elasticity. At the end of both branches are measuring cylinders
that measure the amount of fluid that has flowed in the elastic and inelastic system during a
simulated cardiac revolution (systole and diastole).

100
 Fig. 7.4: Model of blood vessel elasticity: comparison of elastic and non-elastic vessels.

Model of blood vessel elasticity

Task:
1. Demonstrate the elasticity of the vessels using the model.
2. Write down the volumes of liquid that flowed through the elastic and non-elastic pipe.

Measured values:

Conclusion:

101
Blood pressure

The pressure within the arteries varies all the time due to the activity of the heart.
The energy of the cardiac systole speeds up the blood in arteries (longitudinal pressure
along the blood vessels) which applies to the walls of blood vessels (lateral pressure). It is
known as systemic blood pressure. As was mentioned above, during systole, blood is pushed
to the aorta producing systolic pressure. Part of the kinetic energy is stored in the walls of
the aorta and arteries as potential energy. During diastole, the aorta and arteries regain their
original shape (due to elasticity) and the potential energy transforms back to kinetic energy,
creating diastolic blood pressure and diastolic blood flow. Systolic blood pressure is then
determined as the maximum pressure in the arteries during left ventricular systole and
diastolic pressure as the lowest pressure during cardiac diastole made by the elasticity. Mean
blood pressure is calculated by adding one-third of the pressure amplitude (pressure
amplitude = systolic pressure – diastolic pressure) to the value of diastolic pressure (Fig.
7.5). Blood pressure is usually expressed as a fraction of systolic/diastolic pressure.

Dicrotic
notch

Mean Arterial Pressure

Fig. 7.5: Blood pressure curve.

Of note: The World Health Organization (WHO) recommends giving blood

pressure values in mmHg or kPa. In clinical practice, blood pressure is sometimes given
also in Torrs. (1 kPa = 7.5 mmHg = 7.5 Torr = approx. 10 cm H2O).

Blood pressure measurement

Direct (invasive) method - pressure changes are measured directly from the lumen
of the vessel. In this method, the integrity of the skin and the vessel wall is disrupted, where
a catheter filled with a heparinized physiological solution is inserted into the vessel (most
often the arteria subclavia or arteria femoralis). Heparin is a substance that prevents blood
coagulation. The pressure is transmitted by the fluid in the catheter from a place of
measurement to a pressure sensor (electromanometer). The electromanometer transforms
(transduces) mechanic energy (pressure wave) to an electric signal recognizable by
102
computers or other electronic devices. The transducer head can operate on the strain gauge,
capacitive or inductive principle (for details see chapter 15 Biocybernetics – converters for
sensing non-electric quantities). The direct method of measuring blood pressure is
considered to be a very accurate measurement method. It is most often used in research in
experimental procedures or in intensive care units in hospitals to monitor the vital functions
of a patient with catheters inserted. Since the procedure is invasive, this method is not used
in normal clinical practice.

Indirect (non-invasive) method - pressure changes are measured within the cuff,
not directly in the vessel. It is considered to be less accurate, but due to its non-invasive
procedure, it is the most widespread in clinical practice. It is commonly used in the diagnosis
of various diseases.

There are two methods to indirectly measure blood pressure:

• palpation method,
• auscultation method (Riva-Rocci method),
• or their combination.

Palpation method

A stethoscope is not used in the classic palpation method so only systolic blood
pressure values can be measured. With the right hand the examiner palpates the pulse on a.
radialis while using the left hand inflates the cuff. The disappearance of the pulse indicates
that the pressure in the cuff has reached higher pressure than the systolic pressure in a.
brachialis (location of cuff) – the cuff completely compressed the artery and thus prevented
the blood from flowing through it. The examiner then increases the cuff pressure by another
3 kPa (approximately 20 mmHg). By gradually opening the valve on the balloon, the air is
released from the cuff, reducing the pressure on the artery. The value of systolic pressure is
determined at the moment when a pulse wave reappears on a. radialis (perceived by
palpation).

Auscultation method

Wrap the cuff around the person's arm (above fossa cubiti). As in the palpation
method, palpate the pulse on the a. radialis, and increase pressure about 20 mmHg above
the pressure, which the manometer showed when the pulse disappeared. Using the
auscultation method, the so-called Korotkov's sound phenomena (Korotkov - Russian
surgeon, 1905) appear. These sound phenomena occur only when turbulent blood flows
through the artery under the cuff. Place the Recklinghausen's cuff in the middle third of the
arm (a. brachialis, usually on the left side) and a stethoscope just below the lower edge of
the cuff. Increase the pressure in the cuff quickly until the blood circulation under the cuff
is completely stopped. Then, by slowly and gently opening the valve on the balloon, let the
pressure drop in the manometer listening to the artery below the cuff. There is no sound at

103
first (no flow), and the artery is completely collapsed. As the pressure drops more in the
manometer, the first short tapping sounds appear indicating a partial blood flow under the
cuff (turbulent flow, Korotkoff sound I). The mark of the manometer at which the first
sounds appear corresponds to the systolic blood pressure. With a further decrease in the
pressure in the manometer, other sounds could be noticed. The sounds become louder, then
dull and they change into murmurs (Korotkoff sound II). Afterward, the murmurs change
into clear sounds; they reach their maximal intensity, which slowly decreases (Korotkoff
sound III). Subsequently, all sounds will disappear (Korotkoff sound IV). The
disappearance of the sounds indicates the free flow of the blood; in other words, at the
moment the sounds disappear, the diastolic pressure in the artery exceeds the pressure in
the cuff. This means that turbulent flow converts back to laminar (Fig. 7.6).
The blood pressure is expressed as a fraction of systolic/diastolic pressure with the
correct unit in kPa, mmHg, or Torr (e.g., BP = 120/80 [mmHg]). Due to the subjective nature
of the aforementioned methods, the accuracy of measurement is 5 mmHg (Torrs). Thus, the
measured values are always rounded, e.g., 122/81 = 120/80 [mmHg].
Standard values of systolic blood pressure range from 12 to 20 kPa (90 – 140 mmHg)
and from 8 to 12 kPa (60 – 90 mmHg) for diastolic blood pressure. According to WHO,
values of systolic blood pressure above 140 mmHg and diastolic blood pressure above 90
mmHg are acknowledged as pathological (hypertension).

Fig. 7.6: Blood pressure measurement using auscultation method.

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Principles of blood pressure measurement

Prior to the examination, the person should avoid smoking (about 2 hours),
consuming caffeinated or alcoholic beverages, and exercising. The examination is performed
in a sitting or recumbent position, with resting at least 10-15 min before the measurement.
The sleeves on the shirt or dress should be loose, so as not to restrict the blood flow through
the limb during the measurement. The room should be quiet, with a reasonable temperature.
Talking to the person and also to third parties if they are present in the room should also be
avoided. The sphygmomanometer (tonometer) faces the examiner, not the examined person.
The patient sits leaning against his back and the measured limb together with the
sphygmomanometer are placed at heart level. Even when measuring at recumbence, we must
respect the position of the cuff and sphygmomanometer, which must be located at the level
of the person's heart (reducing the effects of gravity and hydrostatic pressure).
Because the blood pressure is measured indirectly (using pressure in the cuff), the
width of the cuff significantly affects the measured values. Therefore, it is necessary to use
the appropriate width of the cuff, which is about 12 cm wide for an adult (standard). The
size of the cuff must be considered in children with a small arm circumference and vice versa
in adults with a large arm circumference. The correct size is dependent on the circumference
of an arm (see Fig. 7.7). If this principle is neglected, the falsely high blood pressure can be
determined (e.g., in bodybuilders using a 12 cm cuff) or falsely low blood pressure (e.g., in
young children or newborns when using a cuff designed for adults). The cuff must meet the
following requirements during the measurement: the width of the cuff should be 40% of the
circumference of the measured limb; the length of the cuff should encircle the entire arm and
cover at least 2-thirds of its circumference.

Maximum arm
Age range Width (cm) Length (cm)
circumference (cm)
Newborn 4 8 10
Infant 6 12 15
Child 9 18 22
Small adult 10 24 26
Adult 12 30 34
Large adult 16 38 44
Thigh 20 42 52

Fig. 7.7: Recommended dimensions for NIBP (non-invasive blood pressure) blood pressure cuffs. Values
can slightly differ among various cuff manufacturers.

105
Types of the tonometers used for indirect blood pressure measurement:

Mercury (alcohol) tonometer

The classic type of Riva-Rocci sphygmomanometer consists of a Recklinghausen’s

cuff, a balloon for blowing air into the cuff, and the tonometer with mercury (nowadays
alcohol or gas) scale in mmHg, kPa, or Torr. The cuff is a flat rectangular rubber bag that is
placed in a canvas bag. A rubber tube with a balloon opens into the cuff, which enables it to
regulate the pressure inside. The other end of the rubber tube is connected to the tonometer,
where the height of the mercury or alcohol column indicates blood pressure values (Fig. 7.8
LEFT).

Fig. 7.8: LEFT: Mercury sphygmomanometer. RIGHT: Spring sphygmomanometer.

Aneroid (spring) tonometer

In this tonometer (Fig. 7.8 RIGHT), the mercury column is replaced by a resistance
spring. The other components are identical to the mercury tonometer. The resistance spring
can be damaged by shocks and impacts during handling, and its properties can also change
depending on the temperature, ambient humidity, etc. Therefore, frequent checking of the
measurement accuracy of the tonometer is important.

Digital tonometer

Nowadays, an automatic or semi-automatic digital tonometer is a very popular type

of sphygmomanometer. They are widespread especially in homes and outpatient practices.
However, the World Health Organization (WHO) still prefers the use of a mercury (alcohol)
tonometer as the most accurate indirect method for blood pressure measurement.
Digital tonometers work on the oscillometric principle. Periodically changing
pressure in the artery causes changes in its volume and thus the radius of the vessel. These
changes are transmitted to the surface of the arm and also to the cuff. The change in the
volume of the artery depends on the difference in pressure inside the artery (blood pressure)
and the surface of the artery (cuff pressure). If the pulse in the artery is maximal, the cuff
pressure equals the mean arterial pressure. Systolic and diastolic pressures are then

106
calculated by the tonometer's software. The most common types of digital tonometers are
the arm and the wrist types (Fig. 7.9).

Fig. 7.9: LEFT: Arm sphygmomanometer. RIGHT: Wrist sphygmomanometer.

Blood pressure measurement

Task:
1. Measurement of blood pressure by auscultation method.
2. Measurement of blood pressure using a digital tonometer.

Procedure (auscultation method):

1. Seat the examined person down at a table, and prepare a sphygmomanometer and a
stethoscope.
2. Find the pulse on the radial artery and measure the blood pressure.
3. Record the measured values of the blood pressure and compare them with
physiological values.
Procedure (digital tonometer):
1. Seat the examined person down at a table, and prepare an automatic digital
sphygmomanometer.
2. Measure the blood pressure. Record the measured values of the blood pressure and
compare them with physiological values.

Measured values:

Conclusion:

107
 8 8 Light and it’s properties

Visible light represents electromagnetic radiation (wave) with a wavelength from

400 nm (violet light) to 700 nm (light red) (Fig. 8.2). It spreads in a vacuum with speed of
approximately c = 3.108 m.s-1. In the material environment, the propagation is slower (the
speed of light in the air is considered the same as in a vacuum).

Wave parameters

• Frequency (f) - a physical quantity that indicates the number of repetitions of a

periodic event per unit time, unit is hertz, Hz (s-1).
• Period (T) - a physical quantity that indicates the duration of a periodic event, the unit
is the second, s.
• Wavelength (λ) - a physical quantity that indicates the distance between recurring
periods of waves, the unit is m, mm, nm.
• Amplitude - maximum deviation (value) of the periodic event, unit depends on the
quantity (Fig. 8.1).

Fig. 8.1: Wave parameters.

The light is characterized by:

𝑐
• wavelength – determines the type (the color) of the light, 𝜆 = 𝑓
• frequency - determines the type or color of visible light (Fig. 8.2) and does not depend
on the environment in which the light propagates (photons of a given type are still
photons of a given type). Photons with the same energy and frequency create
monochromatic light (only one color).
• amplitude – the greater the amplitude of a light wave, the brighter it appears.

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 Fig. 8.2: Visible light within the electromagnetic spectrum.

Light propagation is affected by environmental properties, the environment can be:

• transparent - no light scattering (clear glass);
• translucent - there is a partial scattering (changing the direction of propagation) of
light (frosted glass);
• opaque - light is absorbed or reflected;
• optically homogeneous - has the same optical properties everywhere (e.g. the same
refractive index);
• optically isotropic - the speed of light propagation is the same in all directions;
• optically anisotropic - the speed of light depends on the direction of propagation.

Fig. 8.3: The environment transparent, translucent and opaque.

The ratio of the speed of light in vacuum c and the speed in another medium v is called
the refractive index of the medium (n - absolute index). It is a dimensionless quantity that
depends on the wavelength of light (color, i.e. the type of photons):
𝑐
𝑛=
𝑣

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Reflection and refraction of light

The law of reflection expresses the fact that the angle of reflection α' is equal to the
angle of incidence α. The reflected beam lies in the same plane as the incidence beam (Fig.
8.4).
When the ray of light reaches the boundary of two media with different optical
densities (e.g., air and water), it is partially reflected, and partially penetrates the medium,
i.e. it is refracted. During the refraction, the light changes its direction.

Law of reflection

Specular reflection Diffuse reflection

Fig. 8.3: Specular reflection on the smooth surface vs. diffuse reflection on the rough surface.

Snell's law of refraction expresses that the ratio of the sine of the angle of incidence
α to the sine of the angle of refraction β is constant for two different optical environments
and is equal to the inverse ratio of the refractive indices of the two environments n1 and n2
(also the ratio of light propagation velocities v1 and v2).

sin 𝛼 𝑣1 𝑛2
= =
sin 𝛽 𝑣2 𝑛1

When the light passes from an optically denser medium towards a less dense medium
(n1 > n2), the refraction away from the perpendicular occurs (Fig. 8.4a). When the light

110
passes from a less dense medium to a denser one (n1 < n2), the refraction towards the
perpendicular occurs (Fig. 8.4b).

If the refraction of light away from the perpendicular happens and the refractive angle
is β = 90°, the angle of incidence αc is:
𝑛2
𝛼𝑐 =
𝑛1

A B

α α

β
β

β>α β<α

Fig. 8.4: A - refraction of the light away from the perpendicular - from an optically denser medium to an
optically less dense medium, B - refraction of the light towards the perpendicular - from an optically less
dense medium to an optically denser one.

The angle αc (Fig. 8.5) is the critical angle (angle of incidence at which the angle of
refraction is 90°), e.g. for the glass-air interface it has a value of 42°. If α > αc, refraction of
the light cannot occur, all light is reflected and total reflection occurs.
By measuring the critical angle αc using a refractometer, it is possible to determine the
refractive index of various substances. The total reflection of light is used in optical fibers
and reflecting prisms, which direct or change the trajectory of the rays.

α αc α α’

Fig. 8.5: Refraction of the light (left), refraction with critical angle (centre) and total reflection (right).

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Refractometry

Refractometry is a method used to determine the concentration of substances in a

solution by measuring the critical angle since the refractive index is directly proportional to
the concentration. It is used in clinical laboratories to determine protein concentrations in
serum or plasma. It can be used to determine sugar, alcohol, acids, and other substances.
When light passes from an optically denser environment to an optically less dense
environment (n1 > n2), refraction away from the perpendicular occurs. The critical angle αc
represents the largest angle of incidence at which the refraction of light still occurs
(refraction up to 90 °). If the light beam passes from an optically less dense medium to an
optically denser medium (n1 < n2), it refracts towards the perpendicular, while the critical
angle βc is the largest angle at which the rays still penetrate the optically denser medium
(Fig. 8.4).
If the light rays pass from an optically less dense environment at angles in the range α
= 0 ° up to 90 ° to an optically denser environment, the refractive angle of the light rays
changes in the range β = 0 ° to βc. If the angle of incidence α = 90 °, then according to the
law of refraction:
𝑛1 𝑠𝑖𝑛 90° = 𝑛2 𝑠𝑖𝑛 𝛽𝑚

If the interface between the two media is a refractometer prism and a media layer with
an unknown refractive index (serum, solution, etc.), we can use the refractometer eyepiece
to observe changes in the position of the interface (critical angle) between the light the and
shadow (no light comes at angles > βc). We distinguish dry and immersion refractometers
(Fig. 8.6).

Fig. 8.6: Dry (left) and immersion (right) refractometer.

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 Determination of the saccharose concentration using refractometer

Task:
1. Determine the refractive indices for saccharose solutions of known concentration
using a dry or immersion refractometer.
2. Construct the calibration graph of the refractometer from the measured values of the
refractive indices.

Procedure (dry refractometer):

1. Open the refractometer and place 2 – 3 drops of the liquid with
a known concentration on the matte surface of the refractive
prism.
2. Lock the prisms by the screw and place the refractometer in its
regular position (Fig. 8.7), prepare it for the measurement.
3. Find the light-dark boundary in the right eyepiece by turning
the large setting wheel on the left side of the device (Fig. 8.8).
If the visual field is too dark, illuminate it with a suitable setting
of the mirror. A possible colorful spectrum on the light-dark
boundary may be compensated by means of compensating
prisms that are controlled by the setting wheel on the right side Fig. 8.7: Abbe
refractometer in its regular
of the device. position.
4. When the light-dark boundary is sharp, adjust it to the center of
the reticle by turning the left setting wheel. Read the refractive index of the known
concentration in the left eyepiece on the left scale (with precision to 4 decimals – read
the first 3 decimals directly from the scale and estimate the 4th decimal, Fig. 8.9). Dry
both prisms carefully after each measurement. Use the same procedure with the other
known and unknown concentrations.
5. Construct a calibration graph from the determined refractive indices of solutions of
known concentrations. Using the calibration graph determine the unknown
concentration: find the corresponding value of the solution concentration for the
measured refractive index of the solution with the unknown concentration.

Fig.8.8: Light-dark boundary. Fig. 8.9: Scale in the left eyepiece of refractometer.

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Procedure (immersion refractometer):
1. Fill the refractometric cuvette with distilled water (to the mark).
2. Place the cuvette into the holder. Immerse the measuring prism into the cuvette and
carefully fix the whole tubus into the holder.
3. Using the microscrew of the nonius set the border to the nearest division of the scale.
Then read the number of divisions from the scale in the visual field of the telescope
and add the tenths from the cylinder of the nonius.
4. Determine the refractive index of the measured solutions using the determined position
of the boundary and the table.
5. After finishing each measurement clean the cuvette and dry it properly. Clean and dry
also the measuring prism.
6. Repeat the same procedure to determine the refractive indices of the set of solutions
with known concentrations and a solution with unknown concentration.

Table of the recorded values:

Solution 1 Solution 2 Solution 3 Solution 4
Known concentration C [%]:
Refractive index n:
Solution 5
Unknown concentration C [%]
Refractive index n:

Conclusion:

114
Spectrophotometry

Spectrophotometry is a physicochemical analytical method that uses the absorption of

light by the solution under investigation (visible, ultraviolet, and infrared). It makes it
possible to monitor the dependence of absorption on the wavelength of light, i.e. measure
absorption spectra or determine low concentrations of substances.
Electromagnetic radiation with frequency f propagates through the environment in
quanta (photons) with energy E = h.f, where h = 6,626.10-34 J.s is Planck’s constant. The
total radiation intensity (intensity = energy, flux = amount of energy passing through a unit
area per unit time) depends on the energy of individual photons, as well as on their number.
The composition of radiation is affected by its interaction with the substance environment.
Radiation analysis provides us with information about the environment (concentration),
substances, and about objects that interact with light - it reveals energetic changes in
interacting objects.

Types of radiation interactions with matter:

• photon absorption – the energy of the photon is absorbed by the substance. The
absorbed energy is used to increase the energy state of an atom, molecule, or
complex, which leads to excitation,
• photon emission – energy is released as a photon, atom or molecule returns to the
lower energy state – deexcitation (Fig. 8.10).

h.f ∆E=h.f ∆E=h.f

h.f

Fig. 8.10: Schematic representation of the energy absorption of a photon by an atom and the subsequent
emission of a photon.

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Absorption and emission spectrum

The substance may absorb or emit electromagnetic radiation. Accordingly, the

spectrum is divided into:
• absorption spectrum – arises when light passes through a substance (e.g. gas), in
which some wavelengths of light (some photons) are absorbed,
• emission spectrum – forms the wavelengths of electromagnetic radiation that a
substance emits.
The spectrum division according the shape:
• line spectrum – emitting gases and vapors of elements. It contains only some
wavelengths - the so-called spectral lines (energy of transitions between excited
states and possibly the ground state),
• band spectrum – is emitted by large biological molecules. It consists of a large
number of mutually close spectral lines that merge into wide bands (Fig. 8.11),
• continuous spectrum – emitted by some solids and liquids. It arises e.g. in metals
so that the free electrons of the metal can receive or release any energies that combine
with the energy transitions of the atoms and structures of the metal. The continuous
spectrum indicates that arbitrary energy transitions in a very wide area are possible
in the emitting or absorbing object.

The spectrum is most often expressed graphically in the form of a spectral curve as
absorption, emission, or scattering curve. The spectral curve describes the dependence of
intensity (quantity) on the type (quality) of radiation. Radiation intensity expresses e.g. total
energy over time, energy density, or a number of photons. The type of radiation is given by
its frequency, wavelength, or the energy of individual photons.

relative relative
intensity intensity
[%] [%]
LINEAR CONTINUOUS
100 SPECTRUM 100 SPECTRUM

50 50

0 0
wavelength wavelength

Fig. 8.11: Spectrum examples: a) continuous spectrum; b) line spectrum; c) band spectrum; d) absorption
line spectrum (some “lights” energies are “missing” in the detected light passing through a sample); on the
right their graphic representation.

The probability of energy transitions is high in some molecules and low in others -
bands can be observed in certain parts of the spectrum. For proteins and nucleic acids, a
significant absorption region in the UV spectrum, for proteins with a maximum around 280
nm and nucleic acids with a maximum of around 260 nm. Most biological substances do not

116
absorb visible light - they are transparent and must be dyed (e.g. when viewed under a
microscope).
Pigments, like hemoglobin, absorb light even in the visible part because they contain
bonds with metal (e.g., hemoglobin with iron). The maximum absorption of hemoglobin
within the visible range of the spectrum is from 530 to 570 nm, depending on its type and
form. Since a specific substance always corresponds to a specific spectrum, the chemical
composition of the substances can be determined accordingly. The method that examines the
chemical composition of substances based on their spectrum is called spectral analysis. The
basic instrument of spectral analysis is a spectrophotometer (Fig. 8.12). Using a
spectrophotometer, it is possible to determine whether another substance is present in the
sample, thus determining the purity of the substances, or the amount of substance -
determination of concentration. Chemical reactions or chemical changes of substances also
appear in the spectra.

Spectrum measurement

If the light with intensity (I0) hits the sample, part of the light is reflected (IR),
absorbed (IA), scattered (ID), and passed (I).
Using the appropriate light intensities, the quantities are defined:
• reflection coefficient – R = IR / I0,
• transmittance (transmission) – T = I / I0,
• absorption coefficient (absorbance) – A = IA / I0,
• scattering (diffusion) coefficient – D = ID / I0.

We usually assume that the scattering and reflection of the light on the sample can be
neglected. Then the interaction of the light with the sample is described by the quantities of
light intensity (I0), which enters the examined sample; absorbed light intensity (IA) and light
intensity (I), which passed through the sample layer (Fig. 8.13).

Fig. 8.12: Spectrophotometer. Fig. 8.13: Light transmission through the

spectrophotometer cuvette.

117
 The absorption of any electromagnetic radiation in any environment is described by
Lambert's law:
𝐼 = 𝐼0 𝑒 −𝐸 = 𝐼0 𝑒 −𝑑𝜇

where: E - extinction; μ - linear absorption coefficient; d - path length in the sample = sample
thickness.
Since absorption is the most often measured on liquid samples, Lambert's law
“changes“ to the form of Lambert-Beer's law, which expresses the extinction of a substance
dissolved in a solvent:
𝐸
𝐸 = 𝜀. 𝑐. 𝑑; 𝑐 = [gl−1 ]
𝜀𝑑

where: ε - extinction coefficient (for hemoglobin at 540 nm = 0.7 l.g-1.cm-1); c - concentration

of the substance; d - cuvette thickness (1 cm).

Determination of absorption spectrum of hemoglobin

Task:
1. Measure the absorption spectrum of hemoglobin from venous blood and determine its
concentration.

Procedure:
1. The wavelength range for hemoglobin from venous blood is chosen from 500 nm to
600 nm in 10 nm steps (interval between adjacent points of the measured spectrum).
2. Close the opening of the photocell and set the regulator (marked as 0) to zero on the
scale of intensity or to ∞ on the scale of extinction E.
3. Set the appropriate wavelength at which the spectrum is measured (the first
wavelength is 500 nm).
4. Put the cuvette with the solvent (water) into the light of the spectrophotometer,
switch the photocell on, and set the regulator of intensity (marked as 100) to 100%.
5. Change the cuvette with the solvent for the cuvette with a solution of venous blood,
read the value from the scale of extinction E and write it down next to a particular
wavelength.
6. Set the wavelength of another point of the spectrum. Repeat steps 3 – 5 till you reach
600 nm.
7. From the measured values, construct a curve of the dependence of the extinction E
on the wavelength.

118
 Table of recorded values:
Wavelength Wavelength
Extinction E Extinction E
[nm] [nm]
500 560
510 570
520 580
530 590
540 600

Extinction E

1,2

1,1

0,9

0,8

0,7

0,6

0,5

0,4

0,3

0,2

0,1

0
500 510 520 530 540 550 560 570 580 590 600
Wavelength [nm]

Conclusion:

119
 9 9 Sound and ultrasound

Properties of sound

A sound is a mechanic vibration of a medium through which it propagates

(particle displacement) in the form of a wave. Sound is produced by the vibration of any
object. Sound propagates from the point of origin spherically by the waves and needs a
flexible material environment to propagate compared to electromagnetic waves (e.g., light)
that travel in the form of photons. In fluids, a sound propagates by longitudinal waves, in
solids also by transverse waves. It is transmitted as pressure energy (acoustic pressure wave)
to the surrounding environment. When it reaches a sensory organ (ear) it can be heard
(perceived by the brain).
The basic quantities describing the propagation of an acoustic wave through the
environment are frequency, wavelength, period, amplitude, and speed. The speed of a
sound depends on the physical properties of the environment in which it propagates - density,
flexibility, temperature, humidity, etc. (e.g., the speed of sound in 20 °C air equals 343 m/s).
The speed of sound in the air does not depend on air pressure, sound frequency, or sound
intensity.

Parameters characterizing sound:

• pitch – described by a frequency of a sound,
• timbre (tone color) – distribution of harmonic and subharmonic waves in a sound,
• intensity (volume) – described by a density of acoustic power that passes through the
unit area (W.m-2).

Volume is a subjective evaluation of the hearing perception, expressed in the phones

(Ph) units. The human ear is differently sensitive to distinct frequencies – greater sensitivity
is noted in frequency range 0.7–6 kHz with the most significant sensitivity for frequencies
between 3 – 5 kHz with a maximum of around 3700 Hz.
The sound intensity level (L) is used to compare the intensities of the two sounds.
Unit 1 bel (B), which corresponds to a sound intensity ratio of 1:10, is introduced as a unit
of sound intensity level difference. In practice, a unit 10 times smaller called a decibel (dB)
is used. Usually, the intensity of interest is compared to the hearing threshold. In humans,
the hearing threshold was set for reference tone 1 kHz (0 dB) equals 10-12 W.m-2.

Sound intensity can be calculated as:

𝑰
𝐋(𝐝𝐁) = 10 . 𝑙𝑜𝑔10
𝑰𝟎

where, I – acoustic intensity under interest, I0 – hearing threshold.

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 In humans, audible sound lies within the frequencies of 16 Hz – 20 000 Hz.
Frequencies below 16 Hz are known as infrasound, and above 20 kHz as ultrasound.
Humans are able to perceive neither the infrasound nor the ultrasound.

Ultrasonic diagnostic methods

The physical principle of ultrasonography

Ultrasonography is a diagnostic imaging method based on ultrasound waves (i.e.,
frequencies above 20 kHz). It is commonly abbreviated as “USG” or simplified to “sono”.
Ultrasonography uses quite high frequencies (about 1-20 MHz) that none of the speakers is
able to produce. Thus, it is generated by piezoelectric materials – natural crystals such as
quartz or tourmaline, but also by some biological materials such as DNA or bone. Direct
piezoelectric effect (electricity resulting from pressure) means the production of electric
charge in the solid material after the pressure was applied. You can usually encounter this
effect in microphones (your voice vibrates the piezo membrane) or BBQ lighters (a button
with piezo crystal is pressed by a finger to make a spark). The reverse piezoelectric effect
describes mechanic strain after an electric current was applied. Simply said, piezoelectric
material starts to vibrate with the same frequency as the frequency of the electric current.
Thus, by applying the high-frequency AC current (MHz) to the crystal, the ultrasound waves
are generated (Fig. 9.1).

Direct piezoelectric effect Reverse piezoelectric effect

++++++
Mechanic Mechanic
Piezo Piezo + Electric
element element – input
stress stress

––––––

Electric current formation Shape deformation

Fig. 9.1: Piezoelectric effects demonstration.

An ultrasound transducer, a probe used in ultrasonographic machines, contains a

set of piezo crystals for the production of the waves (generated impulse lasts only a couple
of ms). These waves propagate through the tissues reaching an interface (interface of two or
more materials with different acoustic impedances). The wave is partially reflected and
partially continues into the material. Because the transducer is both a transmitter (sender)
and a receiver, the reflected wave (known as echo, Fig. 9.2) will return to the probe hitting
the crystals. These crystals start to vibrate producing the electric signal with the same
frequency as the echo wave. Acoustic impedance (Z) is given as a product of the speed of
the wave (c) and density (ρ) [kg/m3] of the material (Z = c. ρ).
Penetration of ultrasound waves into a tissue depends on its frequency. In general,
the higher the frequency of ultrasound waves, the lesser the penetration into the

121
material and vice versa (actually this rule is applicable to any type of radiation). However,
the higher the frequency, the better the resolution of the final image. Thus, the
frequency directly affects the formation and quality of an image.

Transducer
(sender / receiver)

Fig. 9.2: Schematic representation of USG image generation.

Basic types of probes (Fig. 9.3):

• linear – the probe consists of a number of parallel transducers (linear arrangement),
and the image is rectangular. Uses higher frequencies approximately 5–10 MHz not
suitable for deep visualization. Examples of use: vascular examination, blood
vessels, thyroid gland, breast visualization, thickness measurement of body fat, etc.
• convex – the probe consists of a convex (U-shaped) series of transducers, the image
has the shape of a circular section (curved). Uses slightly lower frequencies
approximately 1–7.5 MHz suitable for deeper visualization, and good for abdominal
application (organ diagnostics).
• phased array – the ultrasonic beam is either electronically or mechanically deflected,
and the image has the shape of a wide circular section. It is typical for its narrow
beam, which expands depending on the frequency to an almost triangular shape. Its
use is typical for cardiac examinations, where neither linear nor convex probes can
be used due to the ribs and air in the lungs.
• others – e.g., micro convex, T-type, biplanar, endocavital, intrarectal, transvaginal.

Fig. 9.3: Basic types of USG probes: linear (A), convex (B), phased (C).

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Types of ultrasonographic views

• A mode (Amplitude mode) – the simplest type. The transducer scans across the line
through a tissue resulting in a one-dimensional view showing only echoes plotted on
a screen. This mode allows to measuring the distance of the interfaces from which was
the wave reflected (Fig. 9.4).

Fig. 9.4: A-mode.

• B mode (Brightness mode) – two-dimensional

view, represents the most common form of
ultrasonic imaging (Fig. 9.5). In B mode, brightness
depends on the amplitude (intensity) of the reflected
wave. The image is created by adding many A-
mode curves together. Thus, each pixel on the
screen (z-axis) represents the amplitude of the echo.
The more intense the echo, the brighter (whiter) the
Fig. 9.5: B-mode.
picture point. This mode can be extended to a 3D
mode using a 2D probe or 3D imaging in real-time known as a 4D mode.

• M mode (Motion mode) – modification of B mode setup, where moving structures are
displayed. It is most commonly used in echocardiography, where along a chosen
ultrasound line, the motion is shown. Can also be used to determine the speed of the
moving tissue.

The echo wave reflected from the interface of two materials that are not moving in
relation to the probe will have the same frequency as the original wave. In the case, that the
interface or an object is moving (e.g., red blood cells in the vessels), the frequency shift in
the reflected wave will occur. According the effect known as Doppler shift, the wave
reflected from an object moving towards to the probe will have a higher frequency, contrary
to the wave reflected from an object moving away from the probe which will have a lower
frequency (Fig. 9.6).

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 Positive Negative
Doppler shift Doppler shift

Blood Blood
flow flow

Fig. 9.6: (a) Doppler shift occurs if source of the sound or the observer (or both) are moving.
(b) The Doppler effect applies in ultrasonography, where e.g. blood movement shall be visualized. Ft –
original wave, Fr – reflected wave.

Based on the frequency of the echo wave, the ultrasonographic machine is able to
add color to the original black and white image. Usually red or yellow color indicates a
positive Doppler shift, and blue or cyan indicates a negative Doppler shift (Fig. 9.7).
Turbulent flow, mixing of the motions to and away from the probe, can be depicted by a
green or turquoise color. The color scale can be adjusted according to special needs and may
vary with USG machines.
Note: Typically, the arteries run along the veins. Blood flows in opposite directions
in them, so also USG machine will assign the opposite color (red and blue). However, it
doesn’t mean, that vein will be always depicted as blue, and the artery as red. By changing
the angle of the probe, suddenly a vein appears as red and an artery as blue!

Fig. 9.7: Picture showing USG of left liver lobe with Doppler coloring. Red color indicates aorta, blue
color inferior vena cava.

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 Measuring the speed of a blood flow in the arteries using Doppler

Task:
1. Display the flow curve of the a. radialis using the bidirectional hand Doppler (Fig.
9.8).
2. Examine the blood flow rates and calculate the vessel resistance index and the vessel
elasticity index.
3. Compare the obtained values with the physiological values.

Procedure:
1. Work individually or in pairs.
2. Apply a small amount of ultrasonographic gel to the end of the
probe or the patient's skin.
3. Place the probe on the skin in the area of the examined artery (a.
radialis). Slowly move the probe until you find the place where
the Doppler sounds are heard best (see the blood flow curve on
the display). The ideal angle of the probe with the patient's skin is
45 - 60 ° (Fig. 9.9).
Fig. 9.8: Hand Doppler.

Fig. 9.9: The procedure for measuring blood flow in the a. radialis.

4. After stabilizing the flow curve (display of at least 3 consecutive peaks; Fig. 9.10),
press the button on the probe to freeze the image (stop recording).

Parameters describing the shape of the

curve pulse wave:
1. systolic onset,
2. maximum systolic pressure,
3. aortic valve closure,
4. end-diastolic pressure.

Fig. 9.10: Blood flow curve.

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5. Press the side button on the device to display the measured values:
S: maximum blood velocity in systole [cm/s] - highest speed recorded during the pulse
wave (maximum height of the curve during systole),
MN: mean blood velocity [cm/s] - expresses the mean value of the blood flow rate
over the period from the onset of systole to the end of diastole of one cardiac cycle
D: blood velocity in diastole [cm/s] – blood flow velocity at the end of diastole,
MIN: minimum blood velocity [cm/s].

6. Calculate using measured values:

The arterial resistivity index IR – assesses the resistance of blood to flow in a vessel:

(𝑆−𝐷)
𝐼𝑅 = (Physiological value: IR < 1)
𝑆

Pulsatility index IP – determines the pulsatility of a vessel (characterizes by rhythmic

pulsation) related to min. and max. blood flow velocity:

(𝑆−𝑀𝐼𝑁)
𝐼𝑃 = (The physiological range of IP is from 4 to 13; with an average of
𝑀𝑁
6.7 depending on the anatomical location of the peripheral
artery)

7. In the conclusion, compare the shape of the measured pulse wave curve to the
physiological curve and also the calculated IR and IP values to the physiological
values.

Table of recorded values:

value unit
Blood flow in systole S=
Mean blood flow MN=
Blood flow in diastole D=
Minimum blood speed MIN=
Heart rate HR=

Calculations:

Conclusion:

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 Tissue perfusion examination using the plethysmographic probe

Task:
1. Examine forefinger perfusion at different temperatures.

Procedure:
1. Attach the plethysmographic probe to the
forefinger of the examined hand (Fig. 9.11).
2. Observe the pulse wave flow curve for
approximately 1 min (Fig. 9.12) and record the
plethysmographic index (PP) and heart rate
(HR) values.
3. Induce the vasoconstriction by placing the
cooling pad in the palm of your hand for 30 Fig. 9.11: Plethysmographic probe attached
to the forefinger.
seconds. Observe the change in blood flow
(Fig. 9.13) and record the values in the table. Remove the cooling pad and observe the
change in blood flow again.

Fig. 9.12: Physiological blood flow curve Fig. 9.13: Reduced blood flow curve

Table of recorded values:

control cooling rewarming

temperature [°C]
PP index [mV/V]
HR – [beats/min.]

Conclusion:

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 Measurement of tracheal diameter and volumetric blood flow

Task:
1. Measure the diameter of the trachea and carotid artery.
2. Visualize the blood flow of the carotid artery using the Doppler and examine the flow
rate.
Procedure:
1. Apply a sufficient amount of ultrasound gel to the jugular notch.
2. Place the ultrasound probe transversely into the jugular notch, and select the correct
angle and pressure of the probe to make the trachea best visible (Fig. 9.14).
3. Measure the inner diameter and thickness of the tracheal wall and write the values in
the table.
4. Rotate the probe sagittally and by moving to the side visualize the carotid artery.
5. Measure the inner diameter of the artery, and the average flow rate, and calculate the
volumetric blood flow.
Wall
thickness

Fig. 9.14: LEFT: Measuring the diameter (d) and thickness of the tracheal wall; RIGHT: measuring the
velocity of blood flow in the vessel using Doppler

𝛱 . 𝑑2
Volumetric blood flow: Q = TAMEAN 4
where:
TAMEAN – time-averaged mean
blood velocity [cm/s]
Table of recorded values:
d – artery diameter [cm]

value unit
Tracheal diameter
Tracheal wall thickness
Artery diameter
Mean blood velocity
Calculations:

Conclusion:

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Effects of sound

Infrasound

Infrasound is defined as an acoustic wave with a frequency lower than 16 Hz (Fig.

9.15). At high-pressure levels, it can be perceived in the form of vibrations. However, in the
animal kingdom, the perception or even production ability was observed in some species
such as elephants, whales, giraffes, hippopotamuses, rhinoceroses, and even pigeons.
Infrasound can be produced as a result of natural processes like storms, thunders,
earthquakes, ocean wave activity, or wind turbulence. Manmade technology can also
produce such waves e.g., wind turbines, air conditioning, and ventilation systems,
compressors, rocket launchings, and marine or jet engines. Low-frequency characteristics of
infrasound make the waves perfect to travel for very long distances (hundreds of kilometers).

Fig. 9.15: Frequency range of sound fields.

Very low frequencies can be perceived only if the intensity is sufficient enough.
However, while humans are unable to hear in the infrasound range, it can be perceived only
as a vibration (tactile disc receptors instead of the cochlear hair cells). It has been found that
if a person is exposed to infrasound, he may have difficulty performing mental work and
also feel discomfort. If the infrasound pressure level exceeds 100 dB, a person suffers from
dizziness, mental fatigue, irritability, nausea, headache, or loss of balance. However, there
is a small sensitive part of the population, which may experience the annoyance of
infrasound starting at 65 dB intensity. The effects are described as general disharmony,
nausea, disorientation, increased fatigue, sleep disorders or drowsiness, and other
combinations of unspecified symptoms. The cerebral cortex is particularly sensitive to
infrasound with a frequency of 7 Hz, which coincides with the alpha wave frequency of the
electroencephalogram (EEG). Exposure to this type of infrasound can prevent clear thinking
or concentration to perform the delegated task.
Levels of 140 dB to 162 dB, depending on the frequency of 2-20 Hz, cause feelings
of pressure and pain in the ear. At these intensities, an inflammatory reaction at the eardrum
can already be observed.
If the intensity is too high, the effects can also be mediated directly by vibrating
organs, tissues, or other body parts. Vibrational resonance occurs when the excitation
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wavelength and geometrical dimension match. So, if a person is exposed for an extended
period of time, strong infrasound can cause internal organs vibrations and internal
hemorrhage, which may lead to pain or even death.

Audible sound

Periodic sounds (e.g., musical

tones) do not cause any harmful effects in
humans unless the intensity level reaches
the pain threshold. Noise is defined as an
“unwanted” non-periodic sound
containing components with different
frequencies and intensities, which causes
an unpleasant or disturbing sensation.
However, the audio noise can be caused
also by mono-frequency sounds
containing one frequency only. Because
these types of sounds never occur in
nature, they are usually very annoying for
humans (e.g., alarm or PC beeping). Thus,
the introduction of technology into all
areas of human activity results in a
permanent increase in the noise in the
working and living environment (Fig.
9.16). Noise can be steady or variable.
Variable noise can be fluctuating,
intermittent, or simply irregular. If its
intensity fluctuates less than 5 dB it is
considered stable. The degree of noise Fig. 9.16: Representative sound levels.
harmfulness is expressed by the so-called
noise levels and classes. The maximum permissible noise levels in the working environment
are given by the type of work activity, e.g., for tough mental work, the maximum permissible
noise level is 50 dB, for less demanding mental work 70 dB, and for physical work without
mental and sensory activity 90 dB. In public facilities and living environments, the maximum
permissible noise levels are lower, e.g., in medical offices it is 40 dB, in hospital rooms 35
dB and in living rooms only 30 dB.
The experience of noise is determined by several factors, its physical properties
(frequency, intensity), character (permanent, intermittent, irregular), duration, and type of
work performed in a noisy environment. Facilities with excessive noise are for example test
rooms for engines and turbochargers, boiler rooms, laundries, garages with pneumatic tools,
chainsaws, etc. The most common consequence of staying in an environment with excessive
noise is hearing damage. This can be acute when damage to the eardrum, middle or inner
ear occurs suddenly and is considered an injury. Chronic hearing damage develops
gradually, initially manifesting as hearing discomfort, which is a reversible hearing loss for
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frequencies, around 4 kHz. Later, the hearing threshold increases for all frequencies and
hearing loss (hypoacusis) develops into deafness.
In addition to hearing loss and tinnitus, prolonged exposure to excessive noise affects
the regulatory mechanisms of the autonomic and central nervous systems. This is manifested
by cardiovascular disorders (heart rhythm disorders, hypertension), gastrointestinal
disorders, and neuropsychiatric disorders (insomnia, fatigue, dizziness). All these disorders,
in their consequences, reduce work performance.

Ultrasound

Ultrasound represents a wide range of mechanic oscillations starting from 20 kHz up

to several hundred GHz. The region of gigahertz frequencies is sometimes referred to as
hypersonic. Ultrasound is produced by magnetostrictive sources (low-frequency
ultrasound produced by changing the shape of a ferromagnetic metal piece within a variable
magnetic field) or piezoelectric sources (high-frequency ultrasound produced by changing
the shape of a crystal by alternating electric current). Low-frequency ultrasound (20 kHz -
100 kHz) is used in ultrasound surgery but also for cleaning the instruments after the surgery
itself. High-frequency ultrasound is used in physical therapy (1 - 3 MHz, higher
intensities), but especially in diagnostics (2 - 40 MHz, low intensities). Hypersonic (1 - 200
GHz) is for example used in acoustic microscopy.
The interaction of an ultrasonic wave passing through a biological tissue depends on
the frequency and mainly on the intensity of this wave. At higher intensities, the interactions
are known as active, resulting in a change in the biological environment, i.e., the biological
effect of ultrasound. Ultrasound is able to affect biological systems at all levels of their
biological organization through its active interactions, resulting in both negative - inhibitory
effects and positive - stimulating effects. The active type of interaction is used in ultrasound
therapy, ultrasound surgery, and laboratory experiments. If the ultrasound intensity is below
the limit of its biological activity (< 0.1 W/cm2), the interactions are known as passive. These
interactions are the basis of ultrasound diagnostic methods.

Ultrasound does not cause ionization and its direct effect on biological systems is
comprehended by three basic mechanisms:
• heating – is caused by the absorption of the acoustic energy in the tissues and its
conversion into heat. The heating of individual tissues depends mainly on their
physical properties and on their vascularization. The greatest warming occurs at the
interface of soft and mineralized tissues (e.g., muscle and bone),
• mechanic effects – caused by the mechanic forces bound to a high frequency of
ultrasonic oscillations. Remember that ultrasound is also a sound that represents a
pressure wave able to cause a mechanic strain or damage.
• cavitation – the formation of oscillating gas bubbles, which are able to form in the
liquid medium during “the vacuum phase” of the ultrasonic wave. The vibration of the
bubbles is radial (increasing and decreasing their volumes) and can lead to their

131
 collapse. This process creates shock waves capable of mechanically damaging the
surrounding biological structure. Cavitation is also a source of free radicals that can
damage biological systems chemically.

Inhibitory effects of the ultrasound

At the macromolecular level, conformational changes occur, especially in proteins

and nucleic acids. The result is their degradation and partial or complete loss of biological
activity (especially in enzymes). At the cellular and tissue levels, the effects of ultrasound
can damage or destroy both plant and animal cells. Structures such as mitochondria and the
endoplasmic reticulum are very sensitive. Changes in the cytoskeletal system especially in
the microtubules have also been demonstrated. At the tissue level, functional and
immunological changes can be observed.

Stimulating effects of the ultrasound

They can be observed at lower ultrasound intensities and only in the cells with high
regenerative activity (epithelial cells, hematopoietic tissue cells). During the ultrasound
wave exposure, they are growing and also differentiate faster. The mechanism of the
stimulating effect of ultrasound has not yet been clearly elucidated. Ultrasound is thought to
affect the permeability of the biological membranes and the molecular interactions
responsible for cell growth.
The use of ultrasound in physical therapy and surgery is based on active interactions,
i.e., also on the biological effects. Conversely, in diagnostic ultrasound applications, the
biological effects must be either completely eliminated or kept to a minimum so that the
patient is not harmed. Current diagnostic systems meet these requirements. Ultrasound
examinations are safe for both pregnant women and newborns.

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 10 10 Optics and microscopy

Imaging using optical systems

Various optical devices such as magnifier, microscope or video camera, are used to
image objects in medicine. Rectilinear light propagation, independence of beam motion,
laws of reflection, and refraction are the basic principles of geometrical optics used for these
devices to operate.

Basic terms of geometrical optics

• Light beam – a directional projection of light energy radiating from a light source.
A straight line indicates the direction of light propagation in the environment.
• Optical system – a complex of interfaces at which the direction of the rays
emanating from the object is changed by reflection or refraction (camera lens, eye,
microscope eyepiece).
• Real image – the image that can be captured on a screen (film in a cinema).
• Fictive image – the image that cannot be captured on a screen (image in a mirror).
• Object space – the space in front of the optical system (mostly on the left).
• Image space – the space behind the optical system (mostly on the right).
• Optical axis – the optical axis is an imaginary line that defines the path along which
light propagates through the system.
• Focal point (F) – The point on the optical axis where the rays converge after passing
through the lens (object/image).
• Focal length (f) – the distance between the focal point and center of a lens.

Imaging using a thin lens

Lenses are transparent objects made from glass or plastic that are ground and polished
or molded to the desired shape. A simple lens, like a magnifying glass, consists of a single
piece of transparent material, while a compound lens, like a microscope, consists of several
simple lenses, usually arranged along one axis. Most lenses are spherically shaped, i.e. their
two surfaces are parts of the spheres. Lenses are classified by the curvature of two optical
surfaces (Fig. 10.1). Each surface can be convex (bulging outwards from the lens), concave
(depressed into the lens), or planar (flat). A lens is biconvex (or just convex) if both surfaces
are convex – typical is converging lens. If both surfaces have the same radius of curvature,
the lens is equiconvex. A lens with two concave surfaces is biconcave (or just concave) –
typical is diverging lens. If one of the surfaces is flat, the lens is plano-convex or plano-
concave depending on the curvature of the other surface. A lens with one convex and one
concave side is convex-concave or meniscus, which is most commonly used as corrective
lenses.

133
 Fig. 10.1: Types of thin lenses.

• Converging (positive) lens – is convex, thicker in the middle, and thinner at the
edges. A collimated beam of light passing through the lens converges to a spot (a
focus F) behind the lens (Fig. 10.2).

Fig. 10.2: Positive (converging lens).

• Diverging (negative) lens – is concave, thinner in the middle, and thicker at the
edges. A collimated beam of light passing through the lens is diverged (spread) (Fig.
10.3).

Fig. 10.3: Negative (diverging lens).

134
Lenses are characterized by focal length or by its inverse value - optical power (D).

Optical power can be expressed:

1
𝜑= [m−1 ]
𝑓

Diopter D [m-1] is a unit of the lens’ optical power, also referred to as dioptric power,
refractive power, focusing power, or convergence power. It describes the degree to which a
lens, mirror, or other optical system converges or diverges light. Converging lenses have
positive optical power D > 0, while diverging lenses have negative power D < 0. When a
lens is immersed in a different (e.g., not air) refractive medium, its optical power, and focal
length change.

Lens equation

The lens equation expresses the quantitative relationship between the object distance
(a), the image distance (a'), and the focal length (f). The equation is stated as follows:

1 1 1
= + ′
𝑓 𝑎 𝑎

where (a) refers to the position of the object and (a’) refers to the position of the image
from the center of the thin lens.

Magnifying glass

The range of eye vision is limited. Details on distant objects cannot be seen because
their images on the retina are too small. On the other hand, observation of small objects is
limited by the accommodation power of an eye, i.e. human eye is not able to focus light from
really close objects (the distance depends mainly on the age). The human eye resolution is
limited to approximately 0.1 – 0.3 mm. Eye resolution refers to the smallest angle of view
(α), at which an eye is able to distinguish 2 points separately (the larger the angle of view,
the more clearly the detail of the object is observable) (Fig. 10.4).

α
(0.1 – 0.3 mm)

Fig. 10.4: Schematic representation of eye resolution - the smallest angle of view (α).

135
 The point appears on the retina of a healthy eye as a small scattering ring. It is
“detected” by the photosensitive cells (cones). Two points can be distinguished from each
other only if there is at least one free light non-stimulated cone on the retina between their
images - scattering rings (Fig. 10.5). Note that this is really working at the spot of sharpest
vision – fovea.

Fig. 10.5: Schematic structure of cones in the fovea of retina. Pictures A’ and B’ are distinguished
from each other because there is one non-stimulated cone between them. Pictures C’ and D’ merged into
one.

If we want to see a tiny object better, we observe it from a shorter distance. It means
that we magnify (increase) the view angle. The larger the angle is the better the details of the
object. This visual angle (α)can be magnified by a suitable optical device such as
magnifying glass, microscope, or binoculars. The magnifying glass is the simplest optical
device made of a converging lens with a focal length smaller than a conventional visual
length, i.e., = 25 cm. The magnifying glass is characterized by angular magnification
(M):

𝛼′
𝑀=
𝛼𝑙

where (α’) represents the view angle at which one can see the object using the
magnifying glass and the view angle (αl) at which one can see the object at the conventional
visual length of 25 cm (Fig. 10.6).

Fig. 10.6: a) Observation of an object without a magnifying glass - angle of view α; b) observation of the
same object with a magnifying glass - the angle of view α' is larger.

136
 We use the magnifying glass (Fig. 10.7) by placing it as close as possible to the eye
and the observed object is placed in the focus of this magnifying glass (or to a smaller
distance). The magnification of the magnifying glass can be measured using an optical
bench. It is mounted at the end of the optical bench and at a distance of l = 25 cm from the
magnifying glass a screen with a millimeter scale is placed. Between magnifying glass and
the screen, there is a scale (a ruler) attached to a slider. When you observe the screen scale
with your left eye (without a magnifying glass) and the ruler scale with your right eye
(through a magnifying glass) at the same time, the images created on the retinas of both eyes
overlap (see practical task).

Fig. 10.7: The image created by magnifying glass.

Measurement of focal length of a thin converging lens

Task:
1. Determine the focal length of a thin converging lens and its optical power.

Procedure:
1. Place the light source with the object on the left side of the optical bench (Fig. 10.8)
and turn on the light source.
2. Place a shade on the right side of the bench. Move the converging lens (L) between
the object and the shade until we obtain a sharp image of the object on the shade.
3. From the optical bench we read the object distance (a - distance of the object from
the lens) and the image distance (a' - distance of the lens from the shade).
4. By using the lens equation calculate focal length (f).
5. From the inverse value of the focal length, we determine the optical power (D).

137
 Light source L Shade
with an object

a a'
Fig. 10.8: Optical bench – schematic picture. 𝑎. 𝑎′
𝑓=
𝑎 + 𝑎′

1
Table of recorded values: 𝐷= [m−1 ]
𝑓 [𝑚]

100
𝐷= [m−1 ]
𝑓 [𝑐𝑚]

1 2 3
Object distance a [m]:
Image distance a' [m]:
Focal length f [m]:

Calculation:

Conclusion:

138
 Measurement of magnification of magnifying glass

Task:
1. Measure the magnification of the magnifying glass by using an optical bench.

ST
sp

B
S
T
sp

Fig. 10.9: Measurement of magnification of the magnifying glass using the optical bench.

Procedure:
1. Adjust the shade ST with the scale at the distance of 25 cm from the magnifying glass
and fix it. Adjust the scale sp (ruler) so that its image is sharp when viewed with one
eye (Fig. 10.9).
2. On the ruler, choose the distance (n - in millimeters), the increase of which we want to
observe in the shade.
3. Find the corresponding number of divisions (N) on the shade scale, which belongs to
(n) divisions of the ruler scale.
𝛼′ 𝑁
4. Calculate the magnification of the magnifying glass using the relation: M = =
𝛼 𝑛
Repeat the measurement three times and calculate the mean value of magnification M.

Table of recorded values:

n N M

Calculation:

Conclusion:

139
Microscope

The microscope is essentially involved in various methods in biology, histology, or

pathological anatomy. A microscope, similarly to the magnifying glass, is an optical device
designed for observing very small structures that are invisible to the naked eye. The
microscope is an optical system of two or more converging lenses.
Microscopic imaging techniques are distinguished according to the type of radiation
(light) coming into the lens (light, polarized light, etc.). According to the way of the system
illumination, light microscopes are divided into:

• Transmitted illumination (Fig. 10.10 LEFT) – common light microscope. It shines

light upward and through the specimen. This illumination type works best with
specimens that are translucent, such as cells, tissues, embryos, zebrafish, or other
small aquatic samples. This method is used to distinguish the morphological
characteristics and optical properties of the observed material.
• Reflected illumination (Fig. 10.10 RIGHT) – it shines light downward and onto the
specimen, enabling you to observe the reflection. This illumination type works best
for opaque specimens, such as rocks, minerals, plants, insects, and ceramics.
• Fluorescent microscopy – uses fluorescence to generate an image. The observed
light is generally produced within the specimen.

Fig. 10.10 LEFT: Light microscope with transmitted illumination system (source of the light is situated
below sample); RIGHT: Light microscope with reflected illumination system (source of the light is situated
above a sample).

140
 Fig. 10.11: Parts of a common light microscope.

The essential parts of the common light microscope

Structural parts (Fig. 10.11):

• Head – carries the optical parts in the upper part of the microscope.
• Base – holds and stabilizes other components of the microscope. It also carries
microscopic illuminators.
• Arm – connects the base and the head with an eyepiece tube. It is also used when
carrying the microscope.

Mechanic parts:
• The adjustment knobs – are used to focus the microscope. There are two types of
adjustment knobs, i.e., fine adjustment knobs and coarse adjustment knobs.
• Stage with stage control – is the section in which the specimen is placed for viewing.
Stage clips hold the specimen slides in place. The mechanic stage allows the control
of slide positions by moving the slides using the mechanic knobs on the stage.

141
 • Eyepiece tube – it’s the eyepiece holder. It carries the eyepiece above the objective
lens. In some microscopes such as binoculars, the eyepiece tube is flexible and can
be rotated for maximum visualization, for distance adjustment.

Optical parts:
• Eyepiece (Fig. 10.12) – also known as the ocular. This is the part used to look
through the microscope. Its standard magnification is 10x. Optional eyepieces have
magnifications from 5x to 30x.
• Objective lens (Fig. 10.12) – is the most important component of the optical
microscope. It is high-power converging lens positioned very close to the specimen.
Its magnification can reach 100x. Various objectives are frequently placed in the
revolver holder, so it is easy to change them.
• Condenser – the lenses collecting and focusing light from the illuminator into the
specimen. They ensure clear sharp images when a high magnification of 400x and
above is used. The higher the magnification of the condenser, the higher the image
clarity. More sophisticated microscopes have an Abbe condenser that has a
magnification of about 1000x.

Illuminating parts:
• Microscopic illuminator – is the microscopes light source, located at the base. It is
used instead of a mirror.
• Diaphragm – it’s also known as the iris. It is found under the stage of the microscope
and its primary role is to control the amount of light that reaches the specimen. High-
quality microscopes have the diaphragm attached to the Abbe condenser.

numerical
Wide Field magnification aperture
eyepiece
bodytube thickness
length of slide
field
of view diameter coverslip

Fig. 10.12: Description of basic marks on eyepiece (left) and objective (right).

Of note: The image created by the objective lens must be as good as possible, so
some optical defects need to be corrected. Corrections are made by combining lenses made
of different types of glass (soda-lime glass or potassium-lime glass) or flint (soda-lead or
potassium-lead glass). Depending on how the optical errors are corrected in the lenses, the
lenses are divided into achromats (red and blue corrections) and apochromats (multi-color
corrections).

142
Imaging using the light microscope

The observed object (↑) is placed near to the object focus (f0) of the objective lens,
but further than the focal length from the center of the objective (Fig. 10.13). The objective
lens displays the object as an image (↓) in the distance (fe) from the eyepiece (relatively far
from the objective). The image of the observed subject is real, magnified, and inverted when
displayed by the objective lens. This image is observed through the eyepiece similarly to a
magnifying glass. The final image in the light microscope observed by the eye is fictive,
magnified, and inverted. The distance between the image focus of the objective and the
object focus of the eyepiece is called the optical interval of the microscope (Δ) (Fig. 10.13).

Fig. 10.13: Principle that enables magnified observation with a biological microscope.

The magnification of a light microscope

The magnification of the light microscope is the product of the magnification of the
objective (Mob) and eye piece (Me) (Fig. 10.14). The total magnification of the microscope
is expressed by the product:
𝑀 = 𝑀𝑜𝑏 . 𝑀𝑒

x = 100 x

Fig. 10.14: The example of 100 x magnification; 10 x magnification of the objectives and 10 x
magnification of the eye piece.

143
Magnification of the objective and eye piece can be expressed:

Δ 𝑙
𝑀𝑜𝑏 = ; 𝑀𝑒 =
𝑓𝑜𝑏 𝑓𝑒

where (Δ) represents the optical interval of the microscope, (l) represents the conventional
visual length 25 cm, (fob) and (fe) represent the focal distance of the objective and eye piece.
Then for total magnification of the light microscope:

∆ . 25
𝑀 = 𝑀𝑜𝑏 . 𝑀𝑒 =
𝑓𝑜𝑏 . 𝑓𝑒

With laboratory microscopes, many different

magnifications can be reached by changing objectives and
eyepieces. Usually, 3 objectives are mounted on a turret for
convenient magnification change (Fig. 10.15). Typical
magnifications are 10x, 20x, and 50x. By changing of an
eyepiece, also magnification will change as well. If 10x
eyepiece is used with the abovementioned objectives, the
available magnifications will be 100x, 200x, and 500x. It
could be assumed that the magnification of the light
microscope could be increased without limits. However, Fig. 10.15: Microscope objective turret.
the image formed by the lens system of the microscope is
never perfectly “sharp” because of imperfections in the lenses (and also physical limits).
These lens imperfections cause a slight blurring or “overlap” of the images resulting in the
degradation of the final image. Thus, increasing the magnification beyond a certain power
is not useful (likely unwanted). The second consideration is the theoretical limit of the
wavelength of visible light while optical images are formed by the interaction of this light
with an object. In order to distinguish the fine details of an object, the object must be larger
than the wavelength of used light. In limited cases, the resolution of a microscope can be
improved by using monochromatic blue or violet light sources, which have the shortest
wavelengths in the visible spectrum. Objects smaller than the wavelength of light cannot
be observed by a light microscope. For higher magnification the shorter “light”
wavelength is necessary to used, e.g., electron microscope.
The range of useful total magnification for an objective or eyepiece combination is
defined by the numerical aperture (NA) of the optical system. The numerical aperture
(dimensionless number) characterizes the range of angles in which the system can
receive or emit light (Fig. 10.16), thus defining a microscope resolution.

144
 Low
Numerical
Aperture

High
Numerical
Aperture

Fig. 10.16: Types of objectives with various aperture angles. Red arrows represent “acceptance” angle α.

Numerical aperture (NA) is calculated as:

𝑁𝐴 = 𝑛. 𝑠𝑖𝑛𝛼

where (n) represents the refraction index of the surrounding environment (between
a sample and an objective lens), (α) is the maximum angle from the objective axis over which
the light from the specimen can be accepted (Fig. 16.14).

The resolution of a microscope (d) representing the shortest distance

distinguishable by the device can be expressed:

𝜆
𝑑=
𝑁𝐴

where (λ) represents the wavelength of light used and (NA) represents the numerical
aperture.
The resolution of the microscope is directly proportional to the wavelength of the
light used and indirectly proportional to the numerical aperture. In order to achieve the
highest possible resolution, it is necessary either to increase the value of the numerical
aperture or to use light of a shorter wavelength λ. There is a minimum magnification
necessary for the observation of details (500 times NA). The maximum useful
magnification is approximately 1000 times NA. Magnifications higher than this value
(empty magnification) will yield no further useful information or the finer resolution of
image details. It will usually lead to image degradation, where increasing magnification
through the eyepiece or intermediate tube lens only causes the image to become more
magnified and blurrier with no corresponding increase in detail resolution and less depicted
area.

145
Phase-contrast microscopy

Contrast represents differences in intensity and/or color of the light from the observed
object. It allows us to see differences in the object structure. Phase contrast microscopy (Fig.
10.17) is a contrast-enhancing optical technique that can be utilized to produce high-contrast
images of transparent specimens, such as living cells (usually in culture), microorganisms,
and thin tissue slices. In effect, the phase contrast technique employs an optical
mechanism to translate minute variations in light phase (timing) into corresponding
changes in amplitude (intensity), which can be visualized and seen as image contrast.

Fig. 10.17: The figure shows a cut-away diagram of a modern upright phase contrast microscope, including
a schematic illustration of the phase contrast optical train. Partially coherent illumination produced by the
tungsten-halogen lamp is directed through a collector lens and focused on a specialized annulus
(labeled condenser annulus) positioned in the substage condenser front focal plane. Wave passing through
the annulus illuminate the specimen and either pass through without deviation or are diffracted and phase
retarded by the structures and phase gradients present in the sample. Not deviated and diffracted light
collected by the objective is segregated at the rear focal plane by a phase plate and focused at the
intermediate image plane to form the final phase contrast image observed in the eyepieces.

The common light microscope allows you to observe only those objects that absorb
(reflect) light differently (colored slides), so the amplitude (intensity) of the light changes.
Native slides (uncolored slides) absorb very little light and are therefore not suitable for
transmitted light observations in a light microscope. However, the microscopic structures of
the specimen differ in their refractive index and the refractive index of the surrounding
environment. The light passing through the specimen structures (optically denser
environment; Fig. 10.18) is phase-shifted relative to the light wave passing through the
surrounding environment.

146
 Physical principle:

Fig. 10.18: Light phase shift when passing through environments with different optical densities.

Very small differences exist between the refractive indexes of the cells and their
surrounding environment and the cytoplasm (Fig. 10.18b) and the cell structures such as the
cell nucleus (Fig. 10.18c). No significant refractive index difference occurs in the coverslip
and surrounding medium (Fig. 10.18a). Phase contrast makes these tiny differences
visible, it “translates” them into differences in intensity that can be visually observed
and recorded. The light waves passing through cell nuclei, cytoplasm, or water, are shifted
(retarded) by small degrees, since these media have slightly different refractive indices (the
higher the refractive index of a medium, the smaller the speed of light in the medium). As a
result, the light that has passed through a cell nucleus lags behind the light that only had to
pass through water (compare Fig. 10.18a and Fig. 10.18c). The lag is called a phase shift.
The light waves in the phase before their entry into the specimen (wavefronts beneath the
coverslip stay that way) split into phase-shifted ones after they have passed through the
various materials having different refractive indices. The human eye cannot see these
phase shifts; therefore, the phase contrast technique uses optical processing to translate
phase shifts into grey tones (Fig. 10.19).

Fig. 10.19: Native specimen (left); specimen with phase contrast (right).

147
Fluorescence microscopy

The fluorescence microscope (Fig. 10.20) uses a physico-chemical process –

fluorescence (type of luminescence) to show organic and inorganic structures.
Luminescence is a process in which a substance (molecule) emits a different quantum
(photon), usually with a longer wavelength, after absorbing a quantum of light (photon). In
fluorescence microscopy, the specimen is illuminated by light with short wavelengths (UV
radiation, laser). This light is absorbed by the fluorophores (Fig. 10.21) – light absorbing
molecules, causing them to reach an excited state and then emit visible light during their
deexcitation. The source of UV radiation is usually a high-pressure mercury lamp.

Fig. 10.20: The fluorescence microscope configuration.

Physical principle:

The sample for fluorescence microscopy must be fluorescent (emitting visible light).
There are several methods of creating a fluorescent sample. The main techniques are
labeling with fluorescent stains (a fluorophore) (Fig. 10.21) or, in the case of biological
samples, the expression of a fluorescent protein. A fluorophore is a fluorescent chemical
compound that can re-emit light upon light excitation.

Fig. 10.21: Fluorescent dyes.

148
 When the tissue is exposed to UV light or laser, the structures (molecules, atoms)
absorb energy and are excited to a higher electron energy level (Fig. 10.22). The excited
states are usually unstable and return to the ground (lower energy) state. The deexcitation
from a higher energy level to a lower one is accompanied by the emission of a photon
(visible light).

Fig. 10.22: Scheme of absorption of high-energy radiation by a substance and subsequent photon emission
– fluorescence.

Fluorescence microscopy imaging (Fig. 10.23) is used to visualize the cytoskeleton,

amino acids, proteins, DNA or to monitor biochemical events in biology, histology,
immunology, immunocytochemistry, etc.

Fig. 10.23: Examples of fluorescence imaging: from left: cell cytoskeleton, viral DNA, kidney epithelial
cells.

Special light microscopes

• Ultraviolet microscopy – the resolution

of the microscope can be improved if
ultraviolet light or a shorter wavelength (λ
< 350 nm) is used instead of visible light.
However, with this method quartz lenses
must be applied, since regular glass
absorbs ultraviolet radiation. The final
image is not visible to the naked eye,
therefore the image is recorded by the
camera, digitalized, and displayed by a Fig. 10.24: Breast tissue innervation (UV
PC monitor (Fig. 10.24). microscopy).

149
 Infrared microscopy – uses light with
wavelengths from 750 to 1100 nm. Infrared light
penetrates the specimen more easily than visible
light, it is possible to observe even coarser opaque
specimens with this method. Infrared light has the
capability to help visualize biomedical samples
without staining, but the wavelengths of such light
make it incompatible with optical microscopes
and require samples to be prepared in a special
way (Fig. 10.25).

Fig. 10.25: Tissue sample dyed by traditional methods (left), tissue types identified with infrared
microscope (right).

• Confocal microscopy – uses LASER as a light source. A specimen is illuminated by

LASER which causes a light reflection from the specimen. The reflected light passes
through a half-silvered mirror (a semi-
transparent mirror that transmits only part of
the light spectrum and reflects the rest). The
confocal microscope is designed to accept light only
from a thin slice within the tissue and to reject light
reflected and scattered from other regions. Finally,
the light enters the photomultiplier, where it is
amplified and detected by the detector. The confocal
microscope is used to study the cellular architecture
(Fig. 10.26), measure intracellular pH, measure
membrane potential, etc.

Fig. 10.26: Immortalized human skin cells.

Fiber optics – endoscopy

Fiber optic probes such as endoscopes, bronchoscopes, or cystoscopes are used for
viewing the internal tracts of the body. The principle of their operation is based on total
reflection (see Chapter 8). Light travelling (Fig. 10.27) in a material of high index of
refraction is totally reflected back into the material if it strikes the material with lower
refractive index at an angle greater than the critical angle (αc).

150
Angle of incidence in glass larger than the Glass fiber core
critical angle αc Cladding (low
(high refractive index)
refractive index)

Fig. 10.27: Total internal reflection of light in a section of optical fiber.

A typical optical fiber is about 10 µm in diameter and is made of high-purity silica

glass. The fiber is coated with cladding to increase light trapping. Such fibers can carry light
for several kilometers without significant loss.
A fiberscope or endoscope is the simplest of fiber-optic medical devices (Fig. 10.28).
They are used to visualize and examine internal organs such as the stomach, heart, and
bowels. A fiberscope consists of two bundles of optical fibers tied into one flexible unit.
Each bundle is typically a millimeter in diameter consisting of about 10 000 fibers.
According to the application, the bundles can vary in diameter (up to about 1.5 cm) and also
in length (from 0.3 to 1.2 m). Light from a high-intensity source, such as a high-power LED
lamp, is focused into one bundle which carries the light to the organ to be examined. Each
of the fibers in the other bundle collects light reflected from a small region of the organ and
carries it back to the observer. The light is focused on the image which can be viewed on a
screen (Fig. 10.29).

Fig. 10.28: Basic structure of an optical fiber. Fig. 10.29: Upper endoscopy.

Electron microscopy

In the case of electron microscopes – as indicated by their name – the image is

produced by electrons instead of light (photons). An electron microscope focuses the
electrons and uses them to form an image. A high-energy electron beam is produced by an
151
electron gun. For practical reasons of image stability and brightness, the electron microscope
is often operated to give a final magnification of 1 000–250 000 on the screen.

The construction of the electron microscope:

• Electron gun – heated tungsten V-shaped fiber (Fig.

10.30), with a diameter of about 100 μm, heated by
high voltage (20 kV) to a temperature of 2700 K,
which causes the emission of electrons.

Fig. 10.30: The electron gun.

• Magnetic lenses – in the case of common light
microscopy, light can be focused by photon refraction via thin glass lenses. Electrons
cannot be focused that way (they are heavily absorbed by any solid material).
Magnetic lenses are used, where the path of electrons is focused by a magnetic
field (magnetic field curves the path of electron movement). Conventional
magnetic lenses consist of copper coils wound symmetrically around an electron
optical axis. The coil is surrounded by an iron sheath with a narrow gap with a
magnetic field inside (Fig. 10.31).

Fig. 10.31: Magnetic lens and diagram of electron transition through the magnetic field of the lens.

There are two main types of electron microscopes: the transmission electron microscope
(TEM) and the scanning electron microscope (SEM).

Transmission electron microscope

The electrons pass through an ultra-thin section of tissue (60 to 100 nm) inserted
between the condenser and the objective and strike the fluorescent plate or film, where they
create an image visible to the eye (Fig. 10.32, Fig. 10.33). The image (Fig. 10.34) is created

152
on the basis of different electron permeability by individual tissue structures. The resolution
of TEM is up to 0.1 nm.

Fig. 10.32: Transmission electron microscope.

Fig. 10.33: The components of a transmission Fig. 10.34: TEM image showing peripheral
microscope. myelinated fibers and a Schwann cell.

Scanning electron microscope

It is a surface-exploring instrument. The image in a scanning electron microscope

is formed by the impact of primary electrons on the sample surface (Fig. 10.35). Primary
electrons lose a small part of their energy when they hit a solid material due to the elastic
collisions (the surface is typically processed – covered by a thin layer of gold). This happens
mainly during the interaction between electrons and nuclei (there is a deviation of the
electron paths). Nonelastic collisions with weakly bound electrons free some of them from
the electron shell of the atom, these secondary electrons are emitted. Detectors placed above
the specimen capture reflected (secondary) electrons and convert them into a signal (image).
The depth and width of the analyzed sample surface as well as the penetration of primary
electrons itself depend on the value of the acceleration voltage of the electron gun.
153
 Secondary
electrons

X-ray
Primary photons
electrons

Fig. 10.35: Interaction of primary electrons (from electron gun) with sample surface.

Fig. 10.36: Scanning electron microscope.

Fig. 10.37: The components of a scanning electron Fig. 10.38: Human red blood cells in SEM.
microscope.

154
 Measurement by light microscope

Task:
1. Calibrate the eyepiece scale for the given magnification and calculate the calibration
constant.
2. Measure the thickness of a hair or tissue.

Procedure:
1. Set the objective with the smallest magnification.
2. Place the calibration slide with a known scale (e.g., 1 division = 0.01 mm or 1 division
= 0.10 mm).
3. Compare the scale of the calibration slide with the scale in the eyepiece (Fig. 10.39)
and calculate the calibration constant (e.g., there are 3 divisions from the eyepiece in
the 100 µm scale of calibration slide, which means that 1 division is equal to 33.33
µm; 100/3). 33.33 is the calibration constant, and the eyepiece is now calibrated.
4. After the eyepiece is calibrated (do not change the selected magnification of the
objective), insert a hair (Fig. 10.40) on a slide or a histological specimen into a
microscope and calculate the thickness of the tissue according to the number of
corresponding divisions.

Fig. 10.39: Eyepiece calibration – comparison of a eyepiece scale Fig. 10.40: Measurement of a hair
(unknown scale) with a calibration slide scale (known scale). thickness

Calculation:

Conclusion:

155
11 11 Electricity and live organisms

Electric current, voltage, and resistance

Electric current (I) is a stream of electrically charged particles (electrons, ions, holes)
in a conductive medium, or in a vacuum. In a metal conductor, charge transmission is
provided by electrons, in tissues, it is provided by ions or charged colloidal particles. If a
charge (Q) flows through the cross-section of a conductor in time (t), the current (I) is:
𝐼 = ΔQ/Δ𝑡 .
The unit of electric current is ampere [A]. According to the agreement, the direction
of electric current is actually the direction of movement of positive charge particles. The
direction of the electric current in metals is opposite to the direction of the movement of
electrons. (Fig. 11.1)

Fig. 11.1: Direction of electric current vs. movement of the electrons.

There are two main types of electric current:

• direct current (DC) – has constant flow direction of electrically charged particles in
time (electrons or ions in an electrically conductive material). The source of DC is a
battery or an accumulator.
• alternating current (AC) – direction and magnitude are changing over time. The
alternating electric current has frequently a sinusoidal waveform. The source is an
AC generator (rotating magnet in a coil).

The electric voltage (U) represents the difference between the electric potentials (φ)
of two charged (electric) points. The unit of the electric voltage is volt [V].
The following applies to electric voltage:
𝑈 = 𝜑1 − 𝜑2
Note: According to the Slovak technical standard STN 50 160: 2011, the normalized
voltage value in the low voltage network (in the socket) is 230 V and 50 Hz (in the USA: 110
- 120 V, 60 Hz, in Japan: 100 V, 50/60 Hz).

156
 Electric resistance (R) is a physical quantity that expresses the ability of a material to
prevent (resist) the flow of electrically charged particles. The electric resistance is expressed
by Ohm's law (describing how much of “electric force” [voltage] is needed to create the
“electric flow” [current]):
𝑅 = 𝑈/𝐼

The unit of electric resistance is ohm [Ω]. The inverse value of the electric resistance
is called the electric conductivity (G) and its unit is Siemens [S].

Electric resistance depends on:

• material of the conductor – specific electric resistance (ρ), which characterizes the
resistance properties of substances,
• length of conductor – the longer the conductor, the higher the resistance,
• cross-section – the thicker the conductor, the lower the resistance,
• temperature – usually the higher the temperature, the greater the resistance; the
dependence of the resistance on the temperature is influenced by the material of the
substance, e.g., for some substances (Hg at T = 4.15 K), the resistivity at very low
temperatures drops to a non-measurable value - superconductivity.

The electric properties of living tissues are divided into:

• passive electric properties – the behavior of tissues and organs in an electric field,
• active electric properties – electric phenomena that arise from the activity of
excitable tissues (action potential).

Passive electric properties

Conduction of direct current through the tissues

Living tissue is a specific type of conductor. It differs from metal conductors mainly
by macro and microscopic inhomogeneity (different chemical composition, viscosity) and
variable physical properties in both location and time (resistance is not constant). Living
tissue can be characterized as a complex and heterogeneous environment filled with
electrolyte solutions. Unlike metal conductors, tissue electric resistance is variable and
depends on the functional state of the tissue. The carrier of the electric charge is mainly ions
in the biological environment, the conductivity by electrons is practically negligible.

In the biological environment, three mechanisms of electric current conduction apply:

• electrolytic – the movement of ions in solution in an electric field (cytoplasm,
intercellular fluid),
• electrophoretic – the movement of electrically charged colloidal particles (blood),
• electroosmotic – the movement of liquids with different ion concentrations (Fig.
11.2).

157
 Fig. 11.2: Electric current conduction, electrolytic (left), electrophoretic (middle), electroosmotic (right).

Biological tissue contains a number of polar molecules (e.g., H2O). The asymmetrical
distribution of electric charges of the same magnitude and opposite polarity causes that they
manifest as electric dipoles. Since these dipoles e.g., molecules are disordered (Fig. 11.3),
their effects cancel out and the resulting dipole moment equals 0. Therefore, the electric
charge and dipole do not appear externally. However, by the action of the external electric
field (Ee), the electric dipoles are oriented according to the intensity vector of the electric
field (E) inside. Polarization occurs and creates an internal electric field (Ei) in the materials
in the opposite direction. Every substance that polarizes in an external electric field is called
a dielectric (Fig. 11.3).

Fig. 11.3: Disordered dipoles of dielectric (left) and polarization of dielectric (right).

The intensity of the resulting field (E) is less than the intensity of the outer field (Ee)
and its magnitude is │E │=│Ee│ – │Ei│. Corresponding energy losses are converted into
heat, which occurs as an increase in the temperature of a dielectric. The dielectric is generally
characterized by low specific electric conductivity. If the dielectric does not contain free
charge carriers (or contains very few), it is referred to an insulator.
When a DC passes through tissue, it passes through an environment with a different
chemical composition (intercellular space, cell membrane, cytoplasm, organelles, etc.). The

158
resistance related to the DC is called resistance. Each environment is characterized by
specific electric resistance (material constant characterizing the electric conductivity of the
substance):

Specific electric resistance

Tissue
[Ω.m]
cytoplasm and intercellular space 0.2 - 1
cell membrane 106 - 108
body fluids 0.8 - 1.3
muscle tissue 3
adipose tissue 10 - 15
bone tissue 30

Due to the resistance of biological membranes, DC propagates mainly through the

intercellular environment. If the current passes through the membrane, depolarization occurs
and its permeability increases. When the biological membrane breaks down (dead tissue),
the final resistance value is equal to the resistance of the cytoplasm.
The electric resistance of the tissue depends mainly on the functional state of the
tissue. Unlike metal conductors, the resistance of the tissues is not constant over time. After
connecting the electrodes, the tissue resistance decreases rapidly, then the decrease slows
down and stabilizes after 30 to 40 minutes.
The electric resistance of the skin is the easiest to measure in the body. The specific
resistance of the skin is about 1000 times higher compared to the average resistance of other
tissues. The electric resistance of the skin depends mainly on the layer of cornified skin cells,
the humidity, temperature, thickness of the skin, the presence of sweat glands, etc. (Fig.
11.4).

Fig. 11.4: Flow of electric current through the skin layers.

159
 Measurement of the skin resistance

Direct current is used to measure skin resistance. Two non-polarizable electrodes are
attached to the skin. A constant voltage is applied to the electrodes and a change in current
is detected, or conversely, at a constant current, a change in voltage is detected at the
electrodes. A very important factor influencing the measurement of skin resistance is the
transient resistance, which depends on the contact of the electrodes with the skin surface.
Transient resistance can be effectively reduced by degreasing and moisturizing the skin.
The following applies to the total skin resistance: R = Rs + RE1 + RE2 where RE1, RE2
are the transient resistances between the skin and the electrodes and (Rs) is the skin resistance
itself.
A measuring circuit in which the voltmeter is connected in front of the ammeter
according to the wiring diagram (Fig. 11.5) is used.

Task:
1. Determine the skin resistance on dry and degreased and moisturized skin.
2. Compare the measured values for dry and moist skin. Explain why the measured
resistances differs and why the skin resistance decreases with time.

Procedure (dry skin):

1. Set the voltage (U) of the direct current source to 6 V and keep it constant.
2. Attach the metal electrodes to the forearm using rubber bands.
3. Connect the electrodes to a direct current source with a connecting wire.
4. On the multimeter, set the range according to the instantaneous value of the current (I)
to µA or mA. According to the range, the value of the internal resistance (RA) of the
multimeter can be found out. Immediately after the connection of the electrodes, read
the current (I) and write it in the table at the time 0 min.
5. Read the following current values after the first, second, third and fourth minute.
6. Write the measured values in the table and calculate the skin resistance (Rs) using the
given formula.

Procedure (wet skin):

1. Perform the measurements the same way and at the same place on the forearm as for
the dry skin. However, degrease the skin with alcohol and moisten it with the
physiological solution (saline).

𝑈
For skin resistance applies: Rs= − 𝑅A [Ω]
𝐼

where: U – source voltage, I – electric current, RA – internal resistance of the ammeter.

160
 RA
I I RS
A

+
V UR RS
- S

Fig. 11.5: Measurement of skin resistance according to Ohm's law.

Table of recorded values:

Dry skin

Time (min.) RA unit I unit Rs unit

Wet skin

Time (min.) RA unit I unit Rs unit

Conclusion:

161
Conduction of alternating current through the tissues

The tissue resistance to alternating current is called an impedance (Z). Because

both the intra- and extracellular spaces are electrically conductive and the cell membrane
forms an insulator between them, it will act as a biological capacitor when connected to an
AC circuit. The membrane is able to accumulate electricity - it has its capacitance.

Membrane impedance has two components:

• resistance – corresponds to the resistance in the circuit with direct current, does not
depend on the frequency of the alternating current,
• capacitance – the membrane behaves like a capacitor; the capacitance decreases with
increasing AC frequency.

When the biological tissue is connected to an AC, the electric circuit can be described as
a parallel connection of resistor R (tissue electrolyte and membrane channel resistance) and
capacitor C (cell membrane and skin capacity) (Fig. 11.6).

C Z R

Fig. 11.6: Electric model of tissue impedance.

The capacitive component of the conductivity comes from the skin. One plate of the
capacitor is formed by the electrode, the dielectric is formed by the skin itself and the second
plate is a subcutaneous tissue that is well perfused and therefore has a low resistance. All
membranes in the body, including cellular and subcellular, have a significant part in
capacitive conductivity. The higher the frequency of AC, the better the flow of a current
through the membrane (Fig. 11.7). As such, the impedance of the tissue will depend on
the frequency of the AC (Fig. 11.8).

162
 Fig.11.7: Alternating electric current with different frequencies flowing through the tissue. The low-
frequency current passes mainly through the intercellular space (the cell membrane is highly resistant), the
high-frequency current passes through the membrane easily.

Z [ ]

5000
4000
3000
2000
1000
0
f [Hz]
200

10000
100

500

20000
2000
1000

5000

Fig. 11.8: Dependence of impedance on frequency of alternating current.

163
 Measurement of tissue impedance

Task:
1. Measure the whole-body impedance (arm-arm) and its dependence on the frequency
of alternating current (AC).
2. Explain why the whole-body impedance (measured between the upper limbs hand-to-
hand) decreases with increasing frequency of the input voltage.

The whole-body impedance (hand to hand) will be measured using two voltmeters in
the modification according to the Šalanský connection (Fig. 11.8). The frequency of the
input voltage will be changed using the frequency generator (Fig. 11.9).

Procedure:
1. Check the wiring diagram with the teacher. After approval, turn on the AC generator
and set the input voltage UI = 0.2 V. Keep it constant during the whole measurement.
2. The frequency of the alternating voltage is set according to the table in the range from
200 Hz to 4000Hz.
3. Degrease the skin with alcohol and moisten it with saline.
4. Place the clip electrodes on the palmar side of the right and left hand in the first third
of both forearms. Connect the electrodes to the circuit and turn on the multimeters.
5. On the resistance decade, set the resistance (R1) for each frequency to keep a value of
voltage (U1) on the voltmeter (V1) below 30 mV. Subsequently, read the values of (U1)
and (U2) from both voltmeters.
6. Using the measured values, calculate the whole-body impedance (Z).
7. Compile a graph of its dependence on the frequency of the input voltage on a semi-
logarithmic scale using the calculated values of whole-body impedance at individual
frequencies.

𝑈2
Calculate the impedance Z using Ohm's law: 𝑍 = 𝑅1 ( − 1) [Ω]
𝑈1

where: R1 – resistance of the resistance decade, U1 – voltage of the V1 multimeter, U2 – voltage

of the V2 multimeter. Adjustment
rotary button

Frequency
adjustment
Type of the
Output
output wave
voltage

Adjustment
On/Off Activated push buttons
button Channel 1

Fig. 11.9: Frequency generator.

164
Table of recorded values:

f [Hz] 200 500 800 1 000 2 000 3 000 4 000

R1 [Ω]
U1 [mV]
U2 [mV]
Z [Ω]

Z [Ω]
8000

7000

6000

5000

4000

3000

2000

1000

0
100 200 500 800 1000 2000 3000 4000
f [Hz]

Calculations:

Conclusion:

165
Electric current in living tissue
Italian physician and physicist Luigi Galvani observed (around the year 1780) that
the legs of freshly killed frogs were able to move when struck by an electric current. He
believed that he had discovered a certain energetic "fluid" that is part of all living organisms.
Nowadays we already know that irritability or excitability is one of the basic property of
cytoplasmic membranes.

DC and AC electric currents have three effects when passing through living tissue:
• electrolytic – flow of direct current through tissue electrolytes (blood, intercellular
fluid, and cytoplasm) change the chemical composition of electrolytes,
• stimulating – tissue stimulation by rectangular, faradic, or sinusoidally shaped
current,
• thermal effect – heating of tissues, burns.

Electrotherapeutic methods include the use of:

• DC (electrolytic effect – galvanotherapy, iontophoresis),
• low-frequency AC or short pulses of various shapes and durations of DC (stimulation
effect),
• high-frequency sinusoidal AC (especially thermal effect),
• high frequency electromagnetic radiation (thermal effect).

The change in tissue irritability due to the application of electric current is called an
electrotonus. It is a passive propagation of electric charge in excitable cells. The change in
irritability occurs, e.g., when an uninterrupted DC that has no direct stimulating effects is
applied. The stimulation occurs only during sudden changes (pulses) of direct current (and
turning it on and off). However, there is a change in the ion distribution in the environment
under the anode and cathode, which causes changes in the excitability of the membranes.
This effect is mainly used in galvanotherapy. The anelectrotonus corresponds to reduced
irritability under the anode. Anode, the positive electrode attracts negatively charged ions
(anions) from the nerve space (nerve fibers) and increases the membrane potential at a given
location (increased negativity - hyperpolarization on the membrane under the electrode). It
is used in therapy to produce hypoalgesia (mild degree of analgesia - reduced pain
perception). Catelectrotonus, on the other hand, increases irritability under the cathode.
Negatively charged electrode attracts positively charged ions (cations) in the intracellular
space of nerve fibers (the number of cations decreases), thereby reducing the membrane
potential (reducing negativity - depolarization). This partial depolarization increases
irritability.
The electrokinetic motion of ions or solvents in the electric field between the anode
and cathode is called iontophoresis. However, when using high intensities of DC current,
there is a risk of skin burns.
Continuous DC does not have a stimulating effect. In order to effectively stimulate
the tissues, pulses of different shapes and durations are necessary to use. Single pulses are
employed, e.g., in the measurement of chronaxie and for pacing (defibrillation). Faradic
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current is a sequence of low-frequency DC pulses (current) with various duration,
interruption periods, and intensity (length and amplitude).
The irritating effects of low frequency (f) DC pulses and AC (<100 Hz) increase
linearly with the frequency. In the range of 100 - 500 Hz, this increase is less significant.
Between 500 - 3000 Hz, the threshold for stimulation depends on √𝑓 and above 3 kHz there
is a gradual decrease in stimulation effects. With sufficient intensity, it causes muscle tetanus
and dilation of the vascular lumen (vasodilation), which causes an increase in blood flow to
the area, increased resorption, and accelerated absorption of swelling. The most efficiently
stimulating low-frequency AC currents (50 - 100 Hz) are used in electroconvulsive therapy
(electroshock) in patients diagnosed with resistant depression. AC current with a frequency
of 50 Hz and an intensity of approximately 200 mA is applied to the temporal areas, causing
a current flow through the cranial cavity. Sinusoidal waveforms, as well as diadynamic
currents (one-way rectified low-frequency AC currents), are used mainly for their analgesic
and antispasmodic effects, absorption of swellings and hemorrhages, and improvement of
the blood supply to the affected area. Electro stimulation (electro cardioversion) is
employed in the treatment of heart rhythm disorders. A defibrillator can restore normal
heart rhythm during ventricular fibrillation. Usually, a short 5 ms monophasic or biphasic
capacitor produced pulse with voltage up to 5 kV is applied to the chest surface. The
pacemaker is used to stabilize the heart rhythm (adjustment of cardiac arrhythmias) in case
of damage to the heart’s conduction system. A lower voltage pulse is applied directly to the
heart muscle. The shape of the pulse replicates as closely as possible the native pulse
generated by the sinoatrial node.
High-frequency AC currents have no irritating effects as the length of the period is
shorter than the length of the chronaxie. Electrochemical reactions also do not occur. Effects
of the AC currents above 100 kHz are exclusively thermal. Heat is generated directly in the
tissue by dielectric heating (Joule's law), eddy currents, or absorption (Brownian motion).
Thus, the high-frequency diathermy allows the entire volume of tissue to be heated in depth.

Electric shocks and injuries

Electric current passing through the body is considered safe if the person is able to
break free from its effects without assistance. Electric shock is a pathological phenomenon
caused by an electric current flowing through a tissue. The effects of electric current on a
living organism depend mainly on the tissue impedance, the current properties, the time, the
frequency, and the direction of the current flow. AC is generally considered more
dangerous than DC. AC penetrates the tissues and cell membranes easier and has higher
stimulating effects. In addition, high voltage AC is more common in households. Because
people do not have receptors for electric current or voltage, they cannot distinguish whether
the objects or areas are under influence of voltage or electric current. Electric shock usually
occurs when an unprotected human makes a contact (is part of the circuit) with a live electric
component at high voltage and the zero potential of the Earth. Natural phenomena, e.g.,
lightning strike in open space, also cause injuries. The close vicinity of high-power lines or

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high-voltage pylons represents the danger of electric shock as well (the circuit can be formed
even without direct contact – though with the high-voltage component!).
Contact with live conductors:
• single-pole contact – contact of one part of the human body with a live electric part
(phase with voltage) with simultaneous contact with the Earth (also indirectly,
through buildings, etc.). It causes an electric current to pass through the body into
the conductive pad (or through the sole of the shoes) to a place with zero potential.
This is the most common type of electric shock.
• bipolar contact – contact with two electric parts at two different voltages (e.g., two
phases). Electric current is flowing through the affected body areas (e.g., between
both hands).
• non-contact injury – occurs without direct contact of the human body with a live
electric part of the line usually at very high voltages (high voltage lines, transformer
substations, power plants, train or trolleybus lines) or under bad ambient conditions
(e.g., high humidity). Therefore, it is not recommended to approach masts or
overhead lines that have fallen on the ground.

The safe value of direct current is approximately 25 mA. The threshold of perception is
in the range of 3-5 mA. There is a feeling of itching and burning at 5-10 mA. In the range of
20-25 mA contractions appear caused by irregularities in electrochemical gradients as the
current flows through the tissue very unevenly (there is no direct electric stimulating effect
of DC). Tetanus occurs at about 60 mA of DC, at currents of 80-100 mA there is a risk of
respiratory arrest. The safe value for AC is approximately 5-10 mA. But it strongly depends
on the frequency. This situation is demonstrated by Kouwenhoven's graph (Fig.11.10),
which shows the magnitude of AC flowing through the body over time.

Four basic zones are seen:

1. Total safety zone – current perception limit is 0.3 - 0.5 mA.
2. Safety zone – the so-called limit of tolerability, there is a perception of burning,
itching, tingling, or mild involuntary contractions. The affected person can free
himself from the current without assistance.
3. Danger zone – severe electric shocks occur - severe tetanic contractions or burns.
The person may no longer be able to get out of the current without help.
4. Zone of danger (mortality) – respiratory arrest and severe burns occur,
ventricular fibrillations are probable, and the heart can stop working.

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 Interuption of breathing
Area of total safety
Duration of flow of AC (ms)

Sense treshold
Area of safety

Magnitude of AC (mA) c -Fibrilation line

Fig. 11.10: Kouwenhoven graph (I-t graph) for 50 Hz AC current.

The effects and damages caused by electric current depend on the direction and the
areas of the body through which the electric current propagates. The brain and heart are the
most sensitive organs. They generate their own electrical potentials essential for their proper
functioning. The flow of electric current can cause depolarization (excitation) of neurons
and muscle cells and their interconnections, leading to abnormal electrical activity in the
heart and brain. It usually leads to uncontrollable tetanic contractions, respiratory distress,
loss of consciousness, arrhythmias or complete cardiac arrest (asystole), and coma. The most
dangerous injury is therefore considered to be the one produced by the flow of electric
current through the head in any direction. The second most serious is the flow of current
between the right and left hand, or the hands and feet, where the heart and other vital organs
are affected. The least threatening accident is the impact of the current caused by the so-
called step voltage, where the current flows between the right and left foot, which does not
directly endanger the visceral organs. This does not mean, however, that it cannot endanger
the affected person's life.
As the resulting effect of the electric current on the body is time-dependent, it is
necessary to act quickly in the event of an electric shock. However, the health of the rescuer
is paramount, so it is strictly forbidden to touch the stricken person as long as he is still
in contact with a live part of the electric circuit. If possible, pull the wires away from the
affected person or interrupt the circuit by cutting the line. However, be careful never to
touch the circuit with a bare hand, but use non-conductive aids (e.g., a wooden handle
from a broom, a wooden chair, thick rubber gloves)! Then, gently touch the affected
person to verify that he is not under power anymore, then you can start with first aid and/or
resuscitation. Never leave the patient, even if he is conscious. The flow of electric current
can cause disorientation or delusions. Burns caused by the electric current are usually

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whitish, thread-like going through the tissue, which can significantly destroy the tissue (Fig.
11.11). As the current follows the path of the lowest resistance, it usually spreads through
well-perfused tissue to a depth (e.g., between blood vessels, muscles, in the direction of
lymph flow) and not over dry skin with high resistance.

Fig. 11.11: Electrical burn.

In the medical environment, injuries are most common due to improper installation
of electric components or equipment and/or improper use of grounding. Under no
circumstances should be the patient or his bed grounded, especially during defibrillation and
electroconvulsive therapy. When using electric and electronic devices that are in contact
with the patient and under the power of electric distribution, an isolating transformer is
usually used. It separates the “ground” on the primary (electric power) and the secondary
circuit (patient).
Standard protection elements in the electric distribution network (i.e. a standard
socket) are circuit breakers. In the case of an electric failure (e.g., too high current through
the earth conductor, whose threshold level is set) the protection devices are activated, i.e.
they disconnect the electric power (at 230V in time below 0.4 s). Electric current overflow
occurs mainly in the event of a short circuit or contact of conductive material, e.g., person
with a live electric part. These protective components should shut down the power allowing
safe handling of the injured person in case of electric shock. It is worth knowing the color-
codes for electric wires. The left connection of the socket is an active phase (live part), it is
usually black or brown wire. A “return” line (so-called neutral), the right connection of the
socket is usually blue. A yellow-green “earth” wire is connected to the “pin”, which should
be safe even when touched with bare hands (Fig. 11.12).

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 Fig. 11.12: Wiring in electric socket.

Protection is most commonly executed by an appropriate cover (separation and

insulation of live electric parts). The so-called IP protection (ingress protection) expresses
the degree of protection of an electric appliance against the intrusion of a foreign body (first
digit of the code) or liquids (second digit of the code). The maximum possible degree of
protection is IP68. Electric appliances with such a designation are fully dustproof and
waterproof even for permanent immersion in water (Fig. 11.13).

Fig. 11.13: IP rating example.

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12 12 Biophysics of sensory perception – eye, ear

Biophysics of an eye

The optical system of an eye

The eye is a part of a complex optical system – a visual analyzer. The visual analyzer
consists of the eye (cornea, aqueous humor, lens, vitreous humor, retina), optic nerve
(formed by about a million fibers), and the visual cortex. The shape of the eye is given by
the strength of the eye (cornea, sclera) and intraocular pressure (2.0 - 2.9 kPa). The eyeball
has a round shape with a diameter of about 2.5 cm and the optical power approximately
59 D (43D for cornea, 16 – 18 D for lens; D - Diopter = unit of refractory power). In a healthy
(emmetropic) eye, the light rays are bent by the cornea and lens and focused to the point of
sharpest vision (macula lutea). Humans can perceive light in the spectrum of wavelengths
400 - 750 nm. A real, reduced and inverted image of the object is then created on the
retina.
The structure of an eye is analogous to the structure of a camera (Fig. 12.1). The
anterior chamber and lens are similar to a camera lens, the pupil serves as an aperture, and
the posterior chamber and retina are analogous to a photographic emulsion, photographic
plate, or image sensor. Photons of visible light received by the eye are processed by an
optical analyzer and evaluated by the visual center in the occipital lobe.

Retina

Lens
Cornea
Fovea Iris

Pupil
Ciliary muscle
Optic
nerve

Fig. 12.1: Eye - camera analogy.

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Eye accommodation

Accommodation is the ability of an eye to change the optical power of the light
apparatus. Simply said, it serves as a “focus adjustment” to see an object sharp as its
distance from the eye varies. From a physiological point of view, it is a dynamic event in
which the refraction of the lens increases. The lens is suspended on the ciliary muscles and
ligaments, that have the ability to mechanically change the curvature of the lens, and thus
its optical power. When an eye is relaxed (distant viewing), the lens is the least rounded. By
moving an object closer, the ciliary muscles get contracted, while ligaments lose their tension
making a lens round out.

Accommodation ability is defined by two points:

• far point (punctum remotum; PR) – the point located at the furthest distance from an
eye seen sharply when a lens does not accommodate. From a distance of about 5 m,
the lens displays objects without accommodating. Theoretically, for the emmetropic
eye, PR lies at infinity.
• near point (punctum proximum; PP) – the nearest point that is sharply displayed on
the retina when viewed up close. When focusing at a close point, our lens
accommodates the most. With age, as the lens loses elasticity, the accommodation
ability of the eye decreases, and the near point moves away from the eye.

The difference between the distance of the near and the far points is known as
accommodation width (AW). It represents the greatest increase of the light refraction given
by the change of optical power of the lens. AW decreases with age and is calculated as:

AW = PP – PR

Refractive errors of an eye

Refractive errors refer to a group of visual defects caused by errors in the refractive
power of an eye – ametropia. It is an abnormal refractive condition (such as myopia,
hyperopia, or astigmatism) of the eye in which images fail to focus on the retina.
• Spherical ametropia – myopia, farsightedness, senile farsightedness.
• Aspherical ametropia – astigmatism.

Myopia (short-sightedness)

Cause:
• the diameter of the eyeball is greater than 2.5 cm (an eye is too long),
• the refractive power of an eye is greater than 59 D (diameter is normal).

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 Image creation and position of PP and PR:
• rays focused in front of the retina, the image of the object is formed in front of the
retina as well, PP and PR move closer to an eye (Fig. 12.2).

Correction: the person is not able to see distance objects sharply. Biconcave (divergent)
lenses are used as a correction. Laser correction is also possible in some cases.

Fig. 12.2: LEFT: Light beams focusing in front of retina - myopia. RIGHT: Corrected using divergent lens.

Hyperopia (farsightedness)

Cause:
• the diameter of the eyeball is less than 2.5 cm (an eye is too short),
• the refractive power of an eye is less than 59 D (diameter is normal).

Image creation and position of PP and PR:

• rays will focus behind the retina, the image of the object will be formed “behind” the
retina as well, PP and PR move further away from an eye (Fig. 12.3).

Correction: the person is not able to see close objects sharply. Biconvex (convergent)
lenses are used as a correction.

Fig. 12.3: LEFT: Light beams focusing behind retina - hyperopia. RIGHT: Corrected using convergent
lens.

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Presbyopia (old-sightedness or senile vision)

• it is a specific type of hyperopia, also known as “short arm syndrome“,

• typical for people over 45, the elasticity of the lens decreases (dissipation of the water),
refraction power and accommodation ability are reduced, light rays from objects fall
“behind” the retina, PP moves away from the eye,
• correction is the same as in hyperopia – convergent lenses.

Astigmatism

A cornea surface does not have a symmetrical spherical shape (ellipsoid), i.e. cornea
does not have the same curvature (different refraction in the horizontal and vertical planes,
Fig. 12.4). This causes more focal points resulting in blurred vision (Fig. 12.5). Regular
astigmatism is hereditary and irregular one is usually caused by an eye injury. Each eye is
slightly astigmatic (0.25 - 0.5 D). However, this physiological astigmatism does not require
correction.

Fig. 12.4: Vision comparison of emmetropic eye (left) and astigmatic eye (middle and right).

Fig. 12.5: Astigmatic cornea producing two focal points.

Correction: cylindrical lenses (we aim to ensure that the optical power of the correction lens
and the astigmatic eye is the same in the vertical and horizontal direction) or laser surgery
(astigmatic keratotomy) – the procedure of reshaping the cornea for it to be more spherical.

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Scheiner's optometer

A horizontal metal bar with a scale in centimeters contains a revolving metal disk
and two sliders (Fig. 12.6). The examined person is to look through the metal disk and choose
the correct pair of openings. It mainly depends on the light conditions that are related to the
diameter of the pupil. The examined person is supposed to see sharply the needles located
on the sliders.

Fig. 12.6: Scheiner's optometer.

The procedure of determining the near point (PP): looking through the metal disk,
the person focuses on the needle (use only one needle for this procedure). The starting
position for the slider holding the needle is approximately in the middle of the metal bar.
During sliding the needle towards the eye, the person still focuses on this needle with
maximum effort. At one point, the image of the needle will split (maximal accommodation).
Read the distance below the needle, determining the punctum proximum.
The procedure of determining the far point (PR): in the emmetropic eye, PR lies
at infinity. To move PR to the metal bar, it is necessary to use a 4 D converging lens, which
has a focal point of 25 cm. Thus, PR should now lie exactly at a distance of 25 cm from the
examined eye. The starting position of the slider is within the first third closer to the eye.
When carrying out the examination you have to make sure the eye is not accommodated. It
is necessary to look into the distance (not focus on the needle). Start to move the slider away
from the eye, still holding the 4 D lens behind the metal disk. At one point, the image of the
needle will split into two needles. Stop the slider and read the distance below the needle,
determining the punctum remotum.
Then calculate the PP and PR in Diopters. Diopter equals to the inverse value of
focal distance in meters. So, if PP equals 10 cm = 0.1 m, then the calculation will be 1/0.1 =
10 D. To calculate PR, do not forget to subtract the 4 D lens. For emmetropic eye, measured
PR focal distance should lie at 25 cm = 0.25 m and optical power equals to 1/0.25 = 4 D.

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Then PR = 4 D – 4 D = 0 D. Related to given example, accommodation width (AW) will
be calculated as AW = PP – PR = 10 D – 0 D = 10 D.
PP, PR, and AW change in myopic and hyperopic eyes. In the myopic eye, PR is
closer in front of the eye and the difference between PP and PR (AW) is higher. Compared
to the hyperopic eye, PP is further from the eye, and also PR distance increases, thus making
the Diopter value negative. AW becomes smaller.

Example calculation for the myopic eye:

1
PP = 7 cm = =14 D
0.07

= 6 D − 4 D (used lens ) = + 2 D
1
PR =16 cm =
0.16
AW = 14 D − 2 D = 12 D

According to PR, the observed defect has to be compensated by a diverging lens with
the optical power –2 D.

Example calculation for the hyperopic eye:

1
PP = 16 cm = =6 D
0.16

= 2.5 D − 4 D (used lens ) = −1.5 D

1
PR = 40 cm =
0.40
AW = 6 − (−1.5) = 8.5 D

According to PR, the observed defect has to be compensated by a converging lens

with the optical power +1.5 D.

Determination of accommodation width

Task:
1. Find the accommodation width and the near and far points.

Procedure:
1. Determine a near point - punctum proximum (PP) and a far point - punctum remotum
(PR) using Scheiner's optometer.
2. Convert the measured values of PP and PR into diopters (D = 1 / f).
3. Determine the accommodation width (AW) from the difference of PP and PR values
in diopters.

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 Calculation:

Conclusion:

Experiments on Scheiner's optometer

Optic nerves crossing in the chiasma cause the rays falling on one side of the retina
to be perceived on the opposite side of the visual cortex (Fig. 12.7). This can be demonstrated
by experiments with Scheiner’s optometer. Using both needles on the optometer is
necessary. Place the closer needle approximately at a distance of the PP and further needle
slide to the end of the optometer. Focusing on the closer needle will result in split of the
distant one and vice versa.

Fig. 12.7: Information from the right visual field is delivered to the left brain and information from the left
visual field is delivered to the right side of the brain.

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Experiment No. 1
Fix the view on the closer needle (P2 point position; Fig. 12.8), allow the lens to be
accommodated as much as possible. Notice the more distant needle (P1 point position),
which is now split. Because the lens is maximally accommodated, the rays from the double
needle image focus in front of the retina. Thus, the beam from the right side of the duplicate
needle image will hit the retina on the left side. By covering the right hole (which projects
on the left side of the retina), the right needle will disappear, because in the visual cortex,
the image on the left side of the retina is formed on the right side.

Experiment No. 2
Fix the view on the distant needle (P1 point position) and notice the proximal needle
(position of the nearby point P2), which is now split. Because the lens is only slightly
accommodated, the rays from the doubled needle image will fall behind the retina. Thus, the
beam from the right side of the needle image will hit the retina on the right side. By covering
the right hole (which projects on the right side of the retina), the left needle will disappear,
because, in the visual cortex, the image on the right side of the retina is formed on the left
side.

Fig. 12.8: Experiments on Scheiner's optometer. P1 – distant point, P2 – close point.

Stereopsis

Binocular vision is the ability to create a spatial visual perception of a single image.
Basically, it is the creation of a single image in 3D. By fixing a view of a certain object, its
image falls in the place of the sharpest vision (fovea centralis on the yellow spot). The

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images from both eyes merge into a single perception in the visual cortex. Binocular vision
is the basis of spatial vision.

Spatial vision

Sight informs us about the three-dimensionality of the objects and their spatial
relationships with the observer. When looking with both eyes, the movements of the eyes
are performed so that they merge into a unified perception by cortical integration. This
phenomenon occurs when the image of the object is displayed in the same places on the
retina on the so-called corresponding loci. These places on the retina (mainly macula lutea)
have the same spatial value and a common cortical projection in the central optical pathway.
The geometric representation of the corresponding points is the horopter plane,
schematically represented by a circle (Fig. 12.9 RIGHT). All points that are projected into
the horopter area are projected at the corresponding retinal sites, i.e., identical retinal sites
in the right and left eye (point A). These points have a common cortical projection in the
brain. All points that lie outside (point B) or inside (point C) of the horopter's circle are
displayed outside of the corresponding places, on the so-called disparate sites (e.g., the
point B is projected to the right from the central fovea in the left eye L and, on the left in the
right eye R). These points are then perceived as split (physiological diplopia) which is known
as spatial disparity. If the points lie at a small distance from the horopter, they create a
small degree of disparity on the retina and we do not perceive these points twice, but spatially
(spatial stereoscopic vision). If such points lie further or closer to the horopter (B or C) it
is known as spatial dispersion. If they lie within one horizontal plane with A point, but to
the right or left of it, it is known as transverse dispersion.

L R

Fig. 12.9: LEFT: Horopter circle. RIGHT: Example of binocular vision examination.

The right eye watches the object more from the right and the left eye watches it more
from the left – slightly disparate (Fig 12.9 LEFT). You may verify it when you hold the

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finger in front of your nose (at a distance of about 10 cm) and have a look at it alternately by
the left and then the right eye.
Visual acuity

The accuracy of the image depends on the resolution of the eye - visual acuity
(visus). The degree of visual acuity is the minimum separabile. It expresses the eye's ability
to see two close points as separate. This occurs when the rays incoming into an eye from an
object form an angle of at least 1 minute (one degree 1° equals sixty minutes 60'). At this
angle, a line 0.3 mm long is projected into the eye from a distance of 1 m. Two points are
distinguished if the distance between them is at least 73 µm. Thus, two cones are stimulated
by the light and one cone in between them is not (only applies at the place of the sharpest
vision, the resolution elsewhere is lower).

Snellen optotype

Snellen’s (sight testing) chart is used to examine visual acuity. The first optotype was
designed by Herman Snellen in 1862, in the form of a table with special letters to keep a
minimum angle resolution. The chart consists of signs (alphabetical letters, numbers, or
images). Each sign is inserted into an imaginary network of 25 squares. The rays emitting
from two neighboring places enter the eye from a certain distance at an angle of 1 minute
and the rays from the edges of the whole sign at an angle of 5 minutes (Fig. 12.10). The sign
is identified as a whole when it is seen at a visual angle of 5 minutes.

Fig. 12.10: Size of the Snellen's chart character.

Procedure

Snellen optotypes (Fig. 12.11) are designed for a certain distance. Usually of 5 or 6
m (e.g., in the USA it can be 20 feet), which is the minimum distance to exclude
accommodation of the eye. Snellen optotype is hung at the height of the average adult’s
head. Rows of the same size can be illuminated separately from behind. Each line is assigned
a numeric value that contains the distance from which the investigated line should be read.

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 Fig. 12.11: Examples of different types of Snellen's charts.

The examined person stands up on the line drawn 5 meters (or other for which is
designed to) from the Snellen chart. From this distance, the examinee reads the individual
characters from the lines using one eye only. The second eye is softly covered so that no
pressure is exerted on this eye. Examiner switches on the illumination and by pointer points
to the character in that row. The letters, that are pointed to, are chosen randomly to prevent
cheating of the examinee (e.g., insurance fraud, the accusation of the employer, etc.). The
last line that the examinee reads without a mistake determines the result.
The result is recorded in the form of a fraction, where the numerator represents the
distance from which the person is examined (5 m) and the denominator expresses the number
of the distance which is given next to the line that the examined person has read without
error (the distance from which it should be read without any error).

5
Normal visual acuity V = 5 = 1.0
5
Worse visual acuity V = = 0.5
10
5
Better visual acuity V= = 1.25
4

The visual acuity can be worse – the examinee is able to read signs that are supposed
to be read from a distance of e.g. 10 meters. Then, the measured visual acuity is half
compared to normal. Sometimes the visual acuity is better. The examinee is able to read
signs at the distance of 5 m, that are supposed to be read from the distance of e.g. 4 m.

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 Determination of visual acuity

Task:
1. Visual acuity examination training.

Procedure:
1. Work in pairs. The examined person stands up on the line drawn 5 meters from
Snellen’s chart (Fig. 12.12) (minimal distance to exclude accommodation of the lens).
2. The examiner stands up at the chart and gradually switches on the illumination of
particular lines, starting from the largest signs, using a pointer to point at letters in the
line (in irregular sequence).
3. Note the last line in which the examined person read all the signs without any error.
4. Each eye is examined separately, the examined person softly covers another eye (Do
not press on the eye!).
5. The result of the visual acuity determination is expressed by a fraction – the numerator
expresses the distance of the examined person from the chart and the denominator
expresses the number written next to the line that has been the last one correctly read
by the examined person.

Visual acuity evaluation:

5
• Normal visual acuity V = 5 = 1.0
5
• Better visual acuity V = 4 = 1.25
5
• Worse visual acuity V= = 0.5
10

Calculation:

Fig. 12.12: Snellen’s chart.

Conclusion:

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Color vision

The photosensitive part of the human eye is the retina. It contains photoreceptors
rods and cones which are sensitive to light. Before the light beam reaches the
photoreceptors, it must pass through a layer of the nerve fibers, a layer of the ganglion cells,
and a layer of the bipolar nerve cells. Behind the photoreceptors, there is a pigmented layer
that separates the retina from the choroid (Fig. 12.13). Thus, only 10 % of the intensity of
light falling into the eye is used for the excitation of the photoreceptors. The rest of the light
is reflected or absorbed before it hits the photoreceptors.

Fig. 12.13: Layers of the retina in stained tissue (a) and as a schematic (b).

The retina consists of about 7 million cones. They are used during daylight, to
distinguish details and to see colors. Their sensitivity to light differs, reaching a maximum
at a wavelength of 555 nm (yellow-green light). Compared to rods, they are smaller with a
conical shape (Fig. 12.14).
There are about 120 million rods on the retina. They are used for vision at a reduced
light intensity, allowing only black and white (intensity) vision. They are most sensitive to
light with a wavelength of 507 nm (green-blue light). The outer segment of the rod has a
cylindrical shape while the inner segment contains contractile elements capable of varying
the length of the receptor according to the light intensity.
The distribution of photoreceptors on the retina is not uniform. The cones are most
common in the fovea centralis of the yellow spot, where the rods do not occur at all. From
the yellow spot towards the periphery, the number of cones decreases. The rods occur in the
maximum amount in the area about 20° from foveae. Towards the periphery, but also to the
center, their number decreases.

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 Fig. 12.14: Photoreceptors under electron microscope (a) and as a schematic (b).

Types of the vision:

• Scotopic vision – vision at low light intensities, does not distinguish colors – only rods
are activated (monochromatic vision), known as “night vision” (Fig. 12.15),
• Photopic vision – vision under well-lit (bright light) conditions, cones allow color
vision, known as “daytime vision”,
• Mesopic vision – typical for the intensity between dark and light, both rods and cones
are involved in the process, known as “twilight vision”.

Fig. 12.15: Scotopic and photopic vision, mesopic vision forms a continuous transition between the curves.

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Color vision deficiency

Polychromatic light is light consisting of more than one wavelength. Examples of

such light can be sunlight or most of artificial sources (lightbulbs etc.). Contrarily, e.g.
LASER forms a monochromatic light. Decomposition of polychromatic light produce a
color spectrum, consisting of specific colors contained in the light. Colors, based on our
perception, can be divided into basic and supplementary. The basic colors are considered to
be red, green, and blue. Supplementary colors are those that result from the mixing of the
basic colors. Each color is characterized by color tone, brightness, and saturation. The ability
of the human eye to perceive colors is called color vision. The trichromatic theory associated
with the names Helmholtz, Young, and Lomonosov is accepted in clinical practice for the
determination of color vision. The human eye is able to distinguish about 150 colors, which
corresponds to wavelength differences shorter than 3 nm.
The trichromatic theory assumes that the retina contains three types of cones (Fig.
12.16), where each type is most sensitive to each of the three basic color types (known as R,
G, B colors). The same mechanism was used also in artificial display technology (e.g., LCD
screens – Fig. 12.17).
Persons with normal color vision, i.e. those that distinguish all three basic colors, are
called trichromats. Approximately 8% of the male and 0.4% of the female population
experience color vision disorders. Color vision disorders may be partial (-anomaly i.e.
partially disabled) or complete (-anopia i.e. totally disabled). A color-blind person who
perceives only shades of black and white is referred to as monochromat (monochromasia).
Dichromats cannot perceive one of the basic colors. Protanomaly or protanopia is loss or
reduction of the ability to perceive red color. Deuteranomaly or deuteranopia of green
color and tritanomaly or tritanopia of blue color.

Fig. 12.16: Different responsivity of three basic types of cones for blue (Small wavelengths), green
(Medium wavelengths) and red (Large wavelengths). For example, brain perceives “blue”, if only S type of
cones are stimulated, but there is no signal from M and L cones. All other colors result from mixing of the
basic perception of colors by adjusting the intensity of three superimposed light sources generating long,
medium, and short wavelengths. White color consists of all colors together – all three types of cones are
equally stimulated.
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 Fig. 12.17: Trichromacy in the artificial technology of a screen.

Color blindness examination

To test color perception, the pseudoisochromatic plates are used. Different authors
created a specific set of tables, but in practice, the best known are Rabkin's or Ishihara
polychromatic tables. They contain different colorful printed images which show pattern of
numbers, letters or geometric shapes composed of a pattern of differently shaded dots
(circles) in an interchangeable colored background (Fig. 12.18). There are twenty plates, two
of which are intended for methodology demonstration (even a person with a color-deficiency
will be able to distinguish the pattern), ten are for general diagnostics, and eight are for
differential diagnostics. If color perception is impaired, the patient sees shapes that are
invisible to a healthy eye. For example, a person with color vision impairment will see the
number “9” instead of the letter “S” etc.

Procedure
An examined person is requested to sit down turning back towards a window. An
examiner will hold the plates approximately 0.5 – 1 m away and within the same height as
the face of the examinee. Tables must be situated at the vertical plane and thus not be tilted.
Examiner then asks about the specific pattern (number or letter), potentially about the color
of the pattern or background, shown on the plate. The examined person is supposed to tell
the result within a couple of seconds – not able to think too much about a pattern. Examiner
fills up the table with the answers making a conclusion for each plate separately. In the end,

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all plates are evaluated together according to the instructions for the specific table sets
(Ishihara, Rabkin, etc.).

Fig. 12.18: Representative polychromatic plates. On the left side showing number pattern, on the right side
showing geometrical shapes patterns (triangle and circle). The one in the middle is example of plate used to
explain methodology of procedure. This type of the plate is also used to test if the examined person is not
lying (insurance fraud, accusation of the employer, etc.)

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 Examination of color blindness

Task:
1. Practice examination of color perception.

Procedure:
1. Observe the shapes at the test plates and write down the results of the observation
(number or shape) in the table.
2. Evaluate the result of the examination according to the instructions of the plates.
3. In the conclusion, evaluate the result of the examination and state any deviation from
trichromacy.

Table of recorded values:

No. of the No. of the

Finding Finding
picture: picture:
1. 11.
2. 12.
3. 13.
4. 14.
5. 15.
6. 16.
7. 17.
8. 18.
9. 19.
10. 20.

Conclusion:

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Biophysics of an ear

The human ear is the part of the auditory analyzer, in which sound signals are
perceived, transformed and converted into neuronal impulses (action potentials). Humans
are able to perceive the frequencies in the range from 16 Hz – 20 000 Hz. Mechanic waves
with a frequency lower than 16 Hz are called infrasound, while those with a frequency
higher than 20 kHz are known as ultrasound. Our hearing is most sensitive to frequencies
between 3000 – 5000 Hz (with a maximum of around 3700 Hz).
Periodic sounds create tones. A simple tone has a harmonic course and consists of
one frequency only (does not exist in nature), compound tone represents a group of simple
tones or sine waves that are superimposed upon major one (e.g., musical tones). Non-
periodic sounds are perceived in the form of noise (e.g., speech).
Humans are able to hear intensities within the range from 10-12 W.m-2 (corresponding
to a sound intensity of 0 dB) to 10 W.m-2 (which has a sound intensity level of 130 dB) (Fig.
12.19). The lower limit of 0 dB is called the hearing threshold, and the upper limit is
approx. 130 dB as a pain threshold.

10

Hearing threshold

16 20 000

Fig. 12.19: Human auditory field.

Transmission of sound waves through the ear

The ear consists of three parts:

• outer ear (pinna and ear canal) - captures and transmits acoustic signals via the ear
canal to the middle ear. The external ear canal acts as a resonator, which amplifies
frequencies in the range from 2000 to 6000 Hz,
• middle ear is located in the tympanic cavity of the skull. It consists of a tympanic
membrane and three auditory bones (ossicles): hammer (malleus), anvil (incus), and
stirrup (stapes), by which the acoustic impedances are equalized for optimal
transmission of acoustic signals from the air to the fluid of the inner ear. It “amplifies”
the sound, which has lost its intensity (approximately 30 dB) due to the large difference
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 in the acoustic impedances of the air (3.9 kPa.s.m-1) and the inner ear fluid (15,700
kPa.s.m-1). The loss of sound intensity is compensated by 2 mechanisms: 1. conversion
of acoustic waves from a large area of the eardrum (20 mm2) to a small area of the oval
window (2.5 mm2), which represents an increase of the pressure almost 20 times; 2.
the lever system of the middle ear ossicles, where the hammer and the anvil represent
an unequal-arm lever that performs a rotational movement. Another part of the middle
ear is the Eustachian tube, which equalizes the pressures between the tympanic cavity
of the middle ear and the outer environment (actual atmospheric pressure, outer ear).
• inner ear is located in the skull base near middle fossa. This small compartment
consists of hearing and balance apparatus with 2 types of receptors – auditory in the
organ of Corti in the cochlea and vestibular, in the semicircular canals. Sound
perception takes place exclusively in the organ of Corti, which is located on the basilar
membrane in the scala media.

Sound conduction:
• air conduction – the sound is conducted by the air through the ear canal, the middle
ear (by a system of ossicles and a tympanic membrane) to the inner ear,
• bone conduction – acoustic vibrations are transmitted directly to the inner ear by
vibrating the skull bone and “shaking” of the cochlea (Fig. 12.20).

Fig. 12.20: Air (green) and bone (orange) conduction of the sound waves.

Determination of hearing threshold

A person's ability to hear various frequencies is tested by tone audiometry.

Audiometry is an examination method using a tone generator – an audiometer (Fig. 12.21
left). It generates a series of acoustic and vibrational stimuli (tones) with frequencies of 250,
500, 1000, 2000, 4000, 6000, and 8000 Hz with different levels of intensity. The human ear
is differently sensitive to various frequencies. Thus, the aim is to determine the thresholds

191
of hearing for each frequency (the lowest intensity of sound that can be heard by the
examined person).
During air conduction, the examinee is placed in a soundproof room with
headphones on. Each ear is tested separately. The examiner gradually decreases the intensity
(dB) of each simple tone. The person being examined pushes a response button to indicate
that the tone was heard. The audiologist lowers the volume and repeats the sound until the
patient can no longer detect it. This process repeats for each frequency. After all frequencies
have been tested, the second ear can be examined.
During the examination of bone conduction, headphones are replaced by a vibrator
as a wave source. The vibrational motor is placed on the temporal bone (processus
mastoideus). The vibrator shakes the bone (sends the sound wave through the bone) and the
sound is transmitted directly through the bone to the inner ear (cochlea). As with the
headphones, the tones are repeated at varying frequencies and volumes.
The result of the audiometric examination is in the form of an audiogram (Fig. 12.21
right). The audiogram represents the dependence of the sound intensity (in dB on the y-axis)
related to the sound frequency (in Hz on the x-axis). Hearing is considered normal for the
threshold of hearing up to 20 dB. If the threshold of hearing is more than 20 dB, it is
considered a hearing loss or hearing defect.

Fig. 12.21: LEFT: An audiometer. RIGHT: An audiogram.

Tone audiometry can detect 3 basic types of hearing loss:

• Conductive – bone conduction is normal while air conduction has an increased
threshold. It occurs when sound is not conducted efficiently through the outer ear to
the eardrum and ossicles.
• Sensorineural – both air and bone conductions have an increased threshold. It occurs
when there is damage to the inner ear (cochlea), or damage to the neural pathways
between the inner ear and brain.
• Mixed – thresholds of air and bone conduction are increased with more than 10 dB
difference between them. It determines damage in the outer or middle ear and in
the inner ear or auditory nerve.

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 Audiometric examination

Task:
1. Practice an audiometric hearing examination of air and bone conduction.
2. Write down the measured values and evaluate them.

Procedure:
1. A volunteer will be the person being examined.
2. Put on the headphones, taking care of the correct placement: "RIGHT ear" - red,
LEFT ear - blue. Make sure that your hair, jewelry, or glasses do not obstruct the
sound transmission (to prevent incorrect positioning of headphones). A volunteer has
to be concentrated on the test while the rest of the audience must be as quiet as possible.
3. Turn on the audiometer (ask the teacher for assistance) and select the appropriate test
(e.g. PURE TONE), then select the type of audio line: A (air conduction) or B (bone
conduction).
4. Apply frequencies of 250, 500, 1000, 2000, 4000, 6000, and 8000 Hz with different
intensities (dB).
5. The examined person presses the button only upon clearly hearing the tone (do not
press if you only assume hearing the tone). Repeat the examination for bone
conduction using a bone vibrator.
6. Print the resulting audiogram, stick it into the protocol, and describe it.

... stick an audiogram here ...

Conclusion:

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13 13 Electric activity of the heart

Electrocardiography

Electrocardiography (ECG) is a simple noninvasive method to evaluate the electric

biopotentials of the heart. An electrocardiogram represents a graphical electrogram
showing the electric activity of the heart's rhythm measured from the body surface (limbs
and chest). The ECG curve shows only the electric activity of the myocardium, not the
mechanical activity. The depolarizations start at the SA node (sinoatrial node) which acts as
the primary pacemaker generating action potentials by unstable membrane potential. This
phase is called prepotential and replaces the resting membrane potential which occurs in
other types of cells. During atrial systole, the action potential is delayed in the secondary
pacemaker of the AV node (atrioventricular node). After this time, the signal is very rapidly
sent through the septum to the apex through the bundle of His, left, and right bundles branch
into the Purkinje fibers. This conducting system produces the so-called electrical systole
which subsequently triggers the mechanic systole (myocardial contraction).

The typical physiological ECG pattern (Fig. 13.1) consists of:

• isoelectric line – a horizontal line without vertical deviations, which represents
an electrocardiograph running idle or in the time between individual heart
cycles – when a heart is electrically “silent”,
• waves – P, T, U waves are round and slower; Q, R, S waves (or oscillations)
are sharp and faster creating QRS complex. Waves recorded above the
isoelectric line are marked as positive, below the isoelectric line as negative,

Fig. 13.1: Schematic representation of ECG pattern.

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 • segments – short period containing only isoelectric line,
• intervals – the longer section containing waves and/or oscillations.

Atrial QRS complex

depolarization

T wave
P wave U
wave

Ventricular
Ventricular depolarization repolarization

Fig. 13.2: LEFT: Propagation of the electric activity through the atria and ventricles of the heart and
corresponding voltage fluctuations on the ECG record; RIGHT: Duration (s) of intervals and segments of
the physiological ECG curve.

• P wave – represents depolarization of atria, duration 0.08 - 0.11 s (Fig. 13.2).

• PQ segment – (time period from the end of the P wave to the beginning of Q
oscillation) represents the delay (silence) in the AV node, conduction an
excitation from the atria to the ventricles, duration 0.04 - 0.12 s.
• PQ interval – (time period from the beginning of the P wave to the beginning
of Q oscillation) expresses the time duration necessary to transmit an action
potential from atria to the ventricles, duration 0.12 – 0.2 s.
• QRS complex – ventricular depolarization (repolarization of atria is hidden
behind it), duration 0.05 - 0.1 s.
• QT interval – (time period from the beginning of the Q oscillation to the end
of the T wave) represents depolarization and repolarization of the ventricles,
duration 0.35 - 0.44 s.
• ST segment – (time period from the end of S oscillation to the beginning of
the T wave) represents the total ventricular depolarization without electric
activity (plateau phase at the action potential curve), duration 0.12 s.
• T wave – repolarization of ventricles, duration 0.16 s.

ECG leads

The electrodes, placed at the surface of a body (on a skin), measure these electric
potentials to create a graphical recording of the electric activity - an electrocardiogram. An
electrocardiograph is a device that collects the signals. Basically, it works as a voltmeter,
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with one positive and one negative electrode, that records voltage fluctuations to form an
ECG curve (Fig. 13.3). Voltage is formed during the propagation of an electric vector across
the myocardium.

Electrode ECG device

Electrode

Fig. 13.3: Electrocardiograph as a voltmeter.

The speed of a normal ECG recording is usually 25 mm/s i.e., 25 mm on the

electrocardiogram, representing 1 s of time duration in reality. The normal value for the
amplitude of 10 mm/mV is used. These calibrations are then used to measure real durations
and amplitudes of required ECG sections.
Typical is 12-lead ECG, which uses 10 electrodes to measure the electric potentials.
The deposition on the body surface is divided into limb and chest electrodes as follows:

• 4 limb electrodes (Fig. 13.4) – placed at the limbs

(wrists and ankles), they are color-coded: red – right
hand (R), black – right foot (G or N -serves as
grounding), yellow – left hand (L), green – left foot
(F)

Fig. 13.4: Limb electrodes.

• 6 chest electrodes (Fig. 13.5) – attached to the chest,

they are marked V1 to V6 (sometimes C1 to C6):
Midclavicular

V1 – 4th intercostal space, parasternal to the right, line Anterior

auxiliary line

V2 – 4th intercostal space, parasternal to the left, Middle auxiliary

line

V3 – between V2 and V4
V4 – 5th intercostal space in the midclavicular line,
V5 – 5th intercostal space in the anterior axillary line,
V6 – 5th intercostal space in the middle axillary line
Fig. 13.5: Chest electrodes.

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 The body is a reasonably good electric conductor, so it doesn't really matter if you
place an electrode on a shoulder or a wrist, or a thigh or an ankle (Fig. 13.4). However, the
electrodes must form an equilateral triangle, otherwise the ECG curve may be distorted.
The black electrode is grounding; thus, the ECG device does not really need it to
measure the electric vectors. Using 3 limb electrodes (R, L, F) and 6 chest electrodes, the
electrocardiograph produces 12 basic ECG leads: I., II., III., aVL, aVR, aVF, V1 - V6. One
lead is established by two electrodes of the “voltmeter” – one positive and one negative. If
the electric vector is pointing to the positive electrode, a deflection (voltage difference) is
positive. If the electric vector is pointing away from the positive electrode (to the negative
electrode), a deflection is negative.

Leads are generally divided into:

▪ bipolar – the potential difference is gathered from two electrodes (leads: I, II, III, CR,
CL, CF),
▪ unipolar – the potential difference is gathered using only one active electrode and one
indifferent electrode (leads: VR, VL, VF, aVL, aVR, aVF, V1 - V6).

VF VL VR

Fig. 13.6: Limb leads connection.

Specifically:
• bipolar, standard, limb leads (Einthoven's leads) I, II, III (Fig. 13.6):
I. lead – between the right hand (red) and the left hand (yellow)
II. lead – between the right hand (red) and the left foot (green)
III. lead – between the left hand (yellow) and the left foot (green),

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 • bipolar chest leads CR, CL, CF: one active (exploratory) electrode is connected to
the right (R) hand, left (L) hand, or the left foot (F). The second active electrode is
placed at the chest,
• unipolar limb leads VR, VL, VF (v stands for vector) (Fig. 13.6): the potential
difference is gathered from one limb electrode (R, L, F) while the second is indifferent
(Wilson's central terminal),
• augmented unipolar limb leads (Goldberger's leads) aVR, aVL, aVF: the potential
difference is gathered from one limb (R, L, F) electrode while the other two limb
electrodes are added together via two 5 kΩ resistors. This connection leads to an
increase in amplitude by 50% (Fig. 13.7),
• unipolar chest leads V1 - V6 (or C1 – C6): one active electrode is placed in a specific
place on the chest, and the other electrode is indifferent with zero potential (Wilson's
central terminal).

Fig. 13.7: Augmented unipolar limb leads connection.

The term “unipolar” leads to the notion of measuring voltage from one electrode
only, which is a false assumption. Voltage is always measured between two points with
different potentials. Unipolar lead uses the so-called indifferent electrode as a second
electrode, which represents zero potential as a second electrode. Wilson connected 3 limb
electrodes (R, L, F) through 5 kΩ resistors and created the electric “center” of the heart. This
reference electrode is known as Wilson's central terminal (WCT). WCT represents a
negative electrode that is calculated by an ECG machine and lies in the middle of the triangle
according to the 3 limb electrodes (under the AV junction, on the left side of the ventricular
septum). Augmentations in Goldberger's leads are done by adding only two limb electrodes
together to create “zero potential”. Thus, the potential difference is gathered from one limb
electrode and the point of summation of two other electrodes.

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 ECG measurements

Task:
1. Connect the ECG leads and record the ECG curve
2. Calculate the heart rate.
Examine the time duration of the waves and the oscillations.

Procedure:
1. Draw the bipolar limb leads and unipolar chest leads in Figures 1 and 2. Label the
electrodes and leads. Place the ECG electrodes on the patient's body according to the
schematics.

Figure 1:

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 Figure 2:

2. Calculate the average duration of the heart cycle and the heart rate using the average
distance of three RR intervals and the speed of the ECG recording.
3. Mark the isoelectric line, waves and oscillations in the ECG recording and describe
their durations.

... stick an ECG recording here...

Table of recorded values:

value unit length duration unit

Speed of recording: P wave:
distance R1: QRS:
distance R2: T wave:
distance R3: PQ interval:
average: QT interval:

Calculations:

Conclusion:

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The electric axis of the heart

The cardiac electric axis represents the direction of the ventricular depolarization
vector, which arises from the summation of all particular vectors during ventricular
depolarization (Fig. 13.8). Simply put, the electric axis represents how the electric signal
(depolarization) propagates through the ventricles. There is only a weak correlation
between electric and anatomic axes in the frontal plane and no correlation in the transverse
plane! Since the left ventricle is physiologically stronger and bigger (electrically enhanced),
the electric axis will also be primarily to the left.

LV α

RV

Fig. 13.8: LEFT: Schematic representation of the propagation of the depolarization vectors in the left and
right cardiac ventricles (LV and RV); RIGHT: Orientation of the final vector (sum of the vectors) -
orientation of the cardiac electric axis (angle α) in relation to the horizontal plane.

Construction of the cardiac electric axis

The electric axis is expressed only by ventricular depolarization. Thus, the measurement
is done using the QRS complex of the ECG curve. The electric axis of the heart is determined
from the equilateral (Einthoven's) triangle, the sides of which represent the bipolar leads I.,
II., and III. To find the center of the triangle (the starting point of the electric axis of the
heart), run the perpendiculars from the centers of each side. Their intersection indicates the
center of the triangle represents the heart. Using corresponding lead I., II., or III. of the ECG
curve the deviations of the Q, R and S waves are determined. To construct an electric axis,
two out of three leads are sufficient. The amplitudes are measured from the isoelectric line
in mm respecting their signs (Q and S are below the isoelectric line so the values are
negative; R wave runs above the isoelectric line so the value remains positive). Thus, the
total QRS vector deviation is calculated as - Q + R – S (as shown in the example).

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 Example: Lead I. Lead II.

Lead I.: Q = - 5 mm, R = + 25 mm, S = - 10 mm = (-5+25-10) = + 10 mm

Lead II.: Q = - 5 mm, R = + 20 mm, S = 0 mm = (-5+20+0) = +15 mm

The final values are sketched as a distance from the center of the side to the direction
of vertexes corresponding to the resulting value of the vector. From the points on sides of
the triangle, the perpendicular lines are drawn and their intersection determines the final
point of the electric heart axis. The arrow representing the axis vector is then situated
between the center of the triangle and obtained point. To evaluate its inclination, the angle
is measured according to the horizontal plane (Fig. 13.9 LEFT).

Fig. 13.9: LEFT: Construction of electric axis of the heart by an equilateral triangle; RIGHT: physiological
and pathological regions in the x, y coordinate system.

Evaluation

The physiological direction of the heart's electrical axis lies within the range of -30°
to +110° in the horizontal plane (Fig. 13.9 RIGHT). The direction of the axis above +110°
represents pathological deviation to the right, if the angle is below -30°, the deviation is
considered pathological to the left. Excessive deviation to the left can be caused by
hypertrophy of the left ventricle, excessive deviation to the right suggests hypertrophy of the
right ventricle.

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 Measurement of cardiac electric axis

Task:
1. Construct the electric axis of the heart from the ECG recording.
2. Evaluate its direction and orientation according to the picture. Write the conclusion.

Conclusion:

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14 14 Radioactivity and ionizing radiation

Each atom consists of a positively charged nucleus and negatively charged electrons.
There are positively charged protons and electrically neutral neutrons in the atomic nucleus.
The number of protons in the nucleus is equal to the number of orbital electrons. The atom
as a whole is electrically neutral.
Atoms with an excess of energy are in an excited state. If the energy received by the
atom is too high (exceeding excitation), ionization occurs. It is described by the process of
excitation and the process of ionization:
• excitation – an increase in the energy of an atom, nucleus, molecule, or crystal above
its basal energy. After excitation, the system is in the so-called excited state. It can
return to a quiescent (ground) state by emitting energy in the form of a photon,
• ionization – by the gain or loss of one or more electrons from the atom shell, the
atom changes to a positive or negative ion. The electron moves through the
environment with the ability to ionize this environment. For ionization to occur, the
atom must receive sufficient energy – to overcome the energy of ionization potential.
The ionization potential represents tens of eV of energy (for single ionization). The
excess energy turns over to kinetic energy of the ion (or “torn off” electron).

Ionizing radiation represents the high-energy part of the spectrum (Fig. 14.1). It has a
high frequency and a very short wavelength. Ionizing radiation is radiation that transmits
energy in the form of particles (> 10 eV) or electromagnetic waves with a wavelength up to
100 nm or with a frequency > 1015 Hz. It has the ability to directly or indirectly form ions in
the substance through which it passes.

Fig. 14.1: Electromagnetic spectrum.

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Radioactivity

Radioactivity is a significant change in the nucleus of an atom (mass, charge, energy).

The change of the atomic nucleus is governed by certain laws, which have a statistical
character. The change is described by the activity of the sample and the disintegration law
(defining the physical half-life).
Ionizing radiation comes from the nuclei of radioactive elements also known as nuclear
radiation. In addition, there exists also non-nuclear radiation that arises in the electron shell
of an atom (X-rays) or artificially in accelerators of electrically charged particles (electrons,
protons, deuterons, and others). In radioactive processes, the nucleus of an atom is in an
excited and unstable state. The steady state is regained by radiating energy in the form of
particles or photons of electromagnetic radiation.

Two main types of radioactivity are distinguished:

• natural – that occurs in the nuclei of heavy elements from atomic number 84 to the
last naturally occurring element of the periodic table Uranium with atomic number 92.
The largest part of the radioactivity to which humans are exposed to comes from
natural sources, the so-called natural background. The main component of the natural
background is Radon gas, which is formed by the decay of radioactive elements in the
Earth’s crust and as the gas penetrates to the surface. The limit of effective dose for the
population from the natural background is 2-5 mSv / year.
• artificial – that occurs in artificially prepared radioactive nuclei by means of a nuclear
reaction in accelerators or reactors. Their production is aimed at achieving an
imbalance (instability) of the atomic nucleus by the so-called “bombardment” of
nuclei. It should be done by a suitable type of particles that must be absorbed by the
nucleus (the simplest by electroneutral neutrons) to cause an imbalance (change the
stable nuclei into unstable nuclei).

Radionuclide - an unstable form of a chemical element (the same type of atoms that
have the same number of protons, neutrons, and the same energy state, the natural element
is typically a mixture of several nuclides) that is radioactive. Its decay results in the emission
of nuclear radiation. It is also called a radioisotope.
A radioactive substance is any substance that contains one or more radionuclides
whose activity is not negligible from the point of view of radiation protection.

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Types of the ionizing radiation

Directly ionizing radiation

The particles ionize the environment by direct interaction with atoms (molecules).
Directly ionizing radiation is typically formed by charged particles (protons, electrons,
positrons).
Alpha radiation (alpha decay)
Alpha radiation is a stream of positively charged Helium nuclei that move at a speed
of about 20,000 km/s. They are composed of two protons and two neutrons, i.e. they have a
positive electric charge and are considered heavy particles. They cause strong ionization of
the environment (also excitation of molecules and atoms). Their trajectory is mostly straight,
due to their high mass and dense interaction with electrons, their reach (trajectory) is short.
They penetrate a layer of air several centimeters thick or a very thin solid layer. As such,
they cannot penetrate the skin (they are absorbed in the epidermis). They cause damage of
the body by inhalation or through the digestive tract. In the treatment of prostate tumors
nowadays, an alpha radiation is used. The basic principle is the saturation of tumor cells with
boron and subsequent irradiation of it with epithermal neutrons. After absorption, the boron
atom decays and the alpha particle with a range of 9 μm is released.
The alpha decay is described by the formula:
𝑨−𝟒
𝑨
𝒁𝑿 = 𝒁−𝟐𝑿′ + 𝟒𝟐𝜶 e.g. 𝟐𝟑𝟗
𝟗𝟒𝑷𝒖 → 𝟐𝟑𝟓
𝟗𝟐𝑼 + 𝟒𝟐𝑯𝒆

Beta radiation (beta decay)

Beta radiation is represented by particles that are emitted by radioactive nuclei of
elements during beta decay. These particles move very fast (280,000 km/s) and carry
negative (electrons) or positive (positrons) electric charge. Their motion can be easily
affected by an electric field (as their mass and inertia are very low). The ability to penetrate
matter is greater compared to alpha particles and their trajectory is tortuous with less frequent
ionizations (and excitations) during interactions. Beta particles can penetrate only low-
density or low-thickness materials. An air layer of 1 m or a metal plate with a thickness of 1
mm is mostly sufficient to stop them.
Three types of beta decay are distinguished, depending on which particle is emitted by
the nucleus. It is beta plus, beta minus decay, and electron capture.
Beta+ decay: 𝐴𝑍𝑋 → 𝐴
𝑍−1𝑌 + 01𝑒 + 𝜗𝑒
Beta- decay: 𝐴𝑍𝑋 → 𝐴
𝑍+1𝑌
̃𝑒
+ −10𝑒 + 𝜗

Indirectly ionizing radiation

Representatives of indirectly ionizing radiation are photons and especially neutrons,

which do not carry any electric charge (and do not interact with electrons). However, in
addition to indirect ionization, photons also form ions through direct contact with electrons
in atoms.
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Gamma radiation
Gamma radiation is high-energy electromagnetic radiation generated during
radioactive (gamma decay) and other nuclear processes. During a nuclear reaction, the atom
remains in an energetically excited state, and this excess of energy radiates in the form of
one or more photons of gamma radiation during which the nucleus transitions to the lower
energy (e.g. ground) state. The lifetime of the excited nucleus is very short and directly
immeasurable, so the photon is emitted practically at the same time as the original particle.
X-rays
It is non-nuclear ionizing radiation. X-rays are generated in X-ray machines and
particle accelerators during the rapid braking of high-energy electrons in the electron shell.
The energy of the generated radiation depends on the energy of accelerated electrons. In X-
ray machines, the electrons are accelerated by the potential difference (voltage) between the
anode (striking electrode for electrons generating X-rays) and cathode (emitting electrons
usually by high heating).

Other types of ionizing radiation

Cosmic radiation
Cosmic radiation is caused by a stream of particles falling on Earth from space. It
contains mainly protons and some lighter nuclei. Particles originating from space collide
with the nuclei of the elements of the atmosphere in the upper layers of the atmosphere,
creating particles of secondary cosmic radiation. This radiation also contains high-energy
photons.
Proton radiation
The proton is considered a stable, positively charged particle in the atomic nucleus,
but it can decay with a half-life of more than 1035 years. Proton absorption depends on the
energy, it occurs mainly in the so-called Bragg peak (Fig. 14.2) – the energy of proton
radiation is in the range of 200 to 330 MeV. This phenomenon is used in radiotherapy, where
most of the radiation is absorbed in greater depth by the target tissue (tumor) while the
surrounding (healthy) tissue is minimally affected.

Fig. 14.2: Bragg peak – distribution of absorbed proton radiation by tumor.

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Detection and measurement of ionizing radiation

The devices used to determine the presence of ionizing radiation and its physical
characteristics are called detectors of ionizing radiation. These detectors are based on the
common principle, they use the effects of radiation on the environment. The ionizing and
excitatory effects of ionizing radiation are most often used.

According to the principle of detection, the detectors are divided into:

• gas detectors (ionization chamber, Geiger-Müller computer),
• scintillation detectors,
• semiconductor detectors,
• thermoluminescent detectors,
• electronic detectors.

Gas detectors
The principle of detectors is based on gas ionization. The effect of ionizing radiation
generates ion pairs, which are carriers of electric charge. Consequently, the gas becomes
electrically conductive (gases are not electrically conductive under normal conditions). Gas
detectors in medicine are commonly used in the workplaces of radiology, nuclear medicine,
and radiation oncology, where they are used for dosimetry of workplaces.
Ionization chamber
The ionization chamber (Fig. 14.3) is a radiation detector consisting of two insulated
electrodes (anode and cathode), which are placed in a container filled with air or other
suitable gas. A stable voltage of 100 - 1000 V is maintained between the electrodes inside
the chamber. Incident ionizing radiation causes ionization of the gas inside the chamber, the
formation of positively and negatively charged ions. These ions move to the electrodes,
positively charged ions to the cathode, and negatively charged ions to the anode resulting in
a flow of electric charge - an electric current.
This current is measured using a
galvanometer. Ionization chambers are used
in medicine in dosimetry, most often in the
workplaces of radiotherapy and nuclear
medicine. In personal dosimetry, they are
used in the form of a pencil dosimeter to
measure the actual radiation dose that a
person received in an environment with an
ionizing radiation. The dose is evaluated
immediately after the exposure.

Fig. 14.3: The scheme of ionization chamber detector.

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Geiger-Müller (GM) counter
The GM counter (Fig. 14.4) consists of a glass or metal tube. The tube is filled with
inert gas, helium, neon, and argon with a few drops of methyl alcohol or bromine, which
serve as an extinguisher. At one end of the tube in the middle is a metal fiber forming an
anode, most often made of tungsten. At the other end of the tube there is an entrance window
for the radiation. The tube casing is a metal shell - cathode. The potential difference between
the cathode and the anode is approximately 1000 V. Ionizing radiation passes through the
window of the GM tube and causes ionization of the gas inside the tube. Positively and
negatively charged ions are formed, and positive ions are accelerated to the cathode and
negative to the anode. Upon impact of the primary accelerated electrons into other atoms,
these eject electrons from the yet non-ionized atoms. These “secondary” electrons may eject
additional electrons after their acceleration etc. This process is called the avalanche effect.

Fig. 14.4: Scheme of the Geiger-Müller computer.

During the passage of ionizing radiation through the volume of the tube, an electric
discharge is created in the GM tube between the anode and cathode - an electric current that
creates an electric voltage pulse (and a signal on the counter's resistance), which is registered.
The discharge between the cathode and the anode must be "extinguished", quenchers are
used for this, e.g. organic substances (methyl alcohol) or bromine. Their molecules are also
ionized. Due to their size, they have low mobility and create a spatial charge around the
anode, which reduces the potential gradient and interrupts the electric discharge.
Working characteristic and dead time of GM counter
The dependence of the number of impulses per second (registered by the counter) on
the voltage between the anode and cathode for a stable radioactive emitter is called the
working characteristic of the GM counter (Fig. 14.5). The voltage V1 when the counter
starts to register impulses is called threshold voltage. The next part of the characteristic (the
209
range of Ohm’s law) is characterized by a fast increase in the number of impulses with
increasing voltage (as far as the voltage V2). Further increasing of voltage raises the number
of impulses only a little. It is a so-called plateau of the GM counter. The length of the plateau
is more than 300 V and its gradient is 2 – 3 %. Working voltage is selected approximately
in the middle of the plateau. Another increase of voltage (further than V3) raises the
characteristic sharply and a permanent discharge may destroy the counter.

Imp / Sec

plateau

V V V Voltage [V]
1 2 3

Fig. 14.5: The curve of working characteristic of G-M counter.

The GM counter has to “recover” for registration of the next particle of ionizing
radiation. It is not able to register another particle during the course (the time) of the
discharge in it. It can register again when the discharge ceases, the ionized molecules and
atoms recombine, and the voltage returns to its initial value. The time interval when the
computer cannot register another particle is called a dead time of the detector (it is below 1
ms for GM). Because of the dead time, the GM counters are less precise for high intensities
of radiation (around 1000 pulses/s). The measured values at such high numbers of impulses
have to be corrected to the dead time of the computer.

Scintillation detectors

Scintillation detectors work on the principle of excitatory effects of ionizing

radiation, they consist of the following parts:
• scintillation crystal
• photomultiplier
• recording device (pulse computer, PC).
Scintillation detectors are used in nuclear medicine, where they are part of diagnostic
equipment, a gamma camera.

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Scintillation crystal
If the ionizing particle passes through certain substances, a light flash with a wavelength
of about 400 nm (luminescence, scintillation) is created in them. We refer to these substances
as scintillators. The intensity of the light flash depends on the type and energy of the nuclear
radiation. The light photon is registered electronically - by a photomultiplier. Solid, liquid,
and gaseous scintillators are distinguished. The most commonly used scintillation crystal in
medicine is NaI (Tl) the thallium-activated sodium iodide. When working with a gamma
camera, we must comply with standard environmental conditions, i.e. examination room
temperature 22-24 °C, otherwise, the scintillation crystal could be damaged.
Photomultiplier
Photomultiplier transforms light flash (stream of photons) into electrons (photo effect).
These so-called photoelectrons multiply their number by accelerating, hitting the next
electrode (dynode), and releasing more electrons from dynodes into the space of the
photomultiplier. The process ensures the conversion of the light signal (light flash generated
in the scintillation crystal) into an electric pulse (Fig. 14.6).

Fig. 14.6: Schematics of scintillation detector.

The photomultiplier consists of 3 types of electrodes, namely photocathode, dynodes,

and anode. The flash of light strikes the photocathode, where photoelectrons are released as
a result of the photo effect. These then go through a system of secondary photocathodes -
dynodes. The dynodes direct the movement of the electrons to the next dynode and the
electric voltage between the dynodes supplies them with the kinetic energy needed for the
secondary emission of electrons from the next dynode. The resulting photoelectrons after
passing through the system of dynodes fall on the anode, where they are registered as an
electric pulse and this is recorded by a recording device such as a pulse counter or computer.
Scintillation detector dead time
The detector dead time is the minimum time that elapses from the registration of one
particle until the time the detector is able to register another particle. The scintillation in the
detector must fade, the detector must deexcite.

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Semiconductor detectors

The semiconductor detector (Fig. 14.7) is basically an ionization chamber where the
gas is replaced by a solid. The passage of a charged particle does not produce an electron
and a positive ion, but an electron-hole pair. The advantage is that the average energy
required to form the electron-hole pair is e.g. in silicon only 3.6 eV compared to gases 20 -
40 eV.
This means that for semiconductors, the energy needed to “release” the electron, and
thus increase the conductivity is significantly lower than in gases. It is even lower in metals
(conductors where the width of the forbidden energy band is negligible). In semiconductors,
the band gap represents the above-mentioned energy of several eV.

Fig. 14.7: Semiconductor detector diagram (left), electron energy diagram in semiconductor detector
(right); if the energy of the ionizing radiation (E) is less than the gap band energy of the material (E G) the
radiation does not cause the formation of electron-hole pairs (the detector does not detect such radiation).

Modern dosimetry uses devices that combine the principles of several dosimeters.
An example can be the so-called electronic personal dosimeters (EPD, Fig. 14.8). These
dosimeters use a combination of a scintillation crystal and a semiconductor detector or PIN
silicon diode to detect radiation.

Fig. 14.8: Electronic personal detector.

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Thermoluminescent detectors

Thermoluminescent detectors (TLDs) (Fig. 14.9) use a phenomenon called

radiothermoluminescence. If the substance has been exposed to ionizing radiation, it absorbs
energy and remains in an excited state. It begins to emit light only when its temperature rises.
TLDs are suitable crystalline substances in which ionizing radiation causes excitation and
capture of electrons in energetically higher states. When heated, the “captured” electrons are
released and return to a stable energy state along with the emission of light. The substance
emits light that total energy is proportional to the energy of the ionizing radiation absorbed
in the substance. The detection of radiated energy is performed by a special reading device
(detector with photomultiplier), which measures the luminous flux emitted during the
heating of the dosimetric material. TLD detectors are used in personal dosimeters. A plate
of lithium fluoride alkali halide crystal serves as the medium.

Fig. 14.9: Thermoluminescent personal dosimeter.

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 Dosimetric examination of the workplace and personal dosimetry

Task:
1. Measure the level of radiation on the surface of a portable lead container with a stored
γ emitter (Cs137). Measure the radiation level on the cover of the container, on the right
and left sides of the container, and write the measured values in the table.
2. Measure the level of radiation of the β emitter (Sr90).
3. Determine the exposure you obtained during the practical session, convert it into a
dose per year, and compare it with the maximum permissible annual dose according
to Slovakia Law No. 99/2018.

Procedure:
1. Set the switch of the GAMMA-SCOUT radiometer to the center position (γ symbol)
for gamma radiation detection. Pressing the ☢ button puts the radiometer into the
standard mode, and it shows you the present radiation in microsievert per hour.
2. Measure the radiation level on the cover of the container, on the right and left sides of
the container. Write the measured values to the table of recorded values.
3. Set the detector to the β+γ measuring position for the measurement of β radiation.
Write the measured values to the protocol.

Table of recorded values:

Radiation level: β [μSv/h]; γ [μSv/h]
Sr90 (β)
Cs137 (γ) container cover
Cs137 (γ) right side
Cs137 (γ) left side

Calculations:

Conclusion:

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Interaction of alpha, beta, and neutron radiation with matter

Interaction of alpha radiation with matter

The alpha particle loses its energy along its linear path by ionization and excitation.
These two phenomena cause approximately equal energy losses of the alpha radiation. The
absorption of alpha radiation depends on the type of absorbent material. Due to the
large energy losses of the alpha particles, the range of the alpha particles is very small. It is
less than 10 cm in air and only 0.1 mm in water or other biological material for 10 MeV.
Protection: paper, leather.

Interaction of beta radiation with matter

The beta particle loses energy as it passes through the material by excitation and
ionization. The ionization capability is 10 times lower than for the alpha particle. It is mainly
because 7000 times lower mass and a half of electric charge. Beta particle easily changes
trajectory (direction), which is several times longer than the range of the alpha particle. Beta
particles penetrate several meters in the air depending on their energy. In addition, the
interaction of beta particles with the material substances results in bremsstrahlung (braking
radiation) and Cherenkov radiation (Fig. 14.10). Bremsstrahlung represents photons
directly produced by a rapid reduction in kinetic energy of the beta electron (positron) during
its interactions with particles of matter. Cherenkov radiation is represented by photons of
electromagnetic radiation with a wavelength at the transition of ultraviolet and visible light.
β+ particles (positrons) have a very short lifespan. After passing through the medium, they
associate with any free electron (annihilation). Two quanta (photons) with an energy of 511
keV are released. These photons propagate in exactly opposite directions. Protection:
aluminum and other metal foil.

Fig. 14.10: Flight of beta particle through the environment.

215
Interaction of neutrons with matter

Neutrons are particles without an electric charge, thus they do not interact with
the electrons. As such the shell of the atom is unaffected by neutrons directly. Neutrons
only interact with atomic nuclei. The neutron interactions with matter depend on their
energies and are highly improbable a neutron must be very close to the nucleus for the
interaction to occur.

The interactions of neutrons with matter include:

• elastic scattering – occurs when a neutron collides with the nucleus, while the
neutron bounces off it and continues in another direction. The nucleus takes up
the energy of the reflected neutron which moves away at a lower speed. If a
neutron collides with a heavy nucleus, it bounces off at almost the same speed and
loses relatively little energy. When a neutron collides with a light nucleus, the
nucleus gains a lot of energy and this process can efficiently slow down neutrons.
• inelastic scattering – during inelastic scattering, the neutron hits the nucleus and
is trapped in it. A compound nucleus is formed, it is unstable and emits a lower
energy neutron together with a gamma radiation photon, which carries the rest of
the energy (Fig. 14.11).
• transmutation – occurs when a compound nucleus (which is unstable and emits
a particle such as a proton, alpha particle, or another neutron) created by the
impact of a neutron changes into the nucleus of another element. This reaction is
likely to occur when the energy of the incident radiation is several hundred eV to
tens of MeV.
• radiation capture – is one of the most common neutron interactions with matter.
The nucleus captures the incident neutron and emits gamma rays. The created
radioisotope is usually a beta or gamma emitter (Fig. 14.11). This reaction is
typical for low-energy neutrons.
• fission reaction – a type of reaction in which a nucleus splits into two nuclei.
Neutron protection: slowing down fast neutrons by interactions in water (the heavy
water D2O – deuterium is formed) and then elimination by light metals (e.g. boron).

Fig. 14.11: The scheme of radiation capture.

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Interaction of gamma radiation with matter

When gamma radiation penetrates a material medium, it is absorbed by three

mechanisms:
• photoelectric effect (photo effect) – arises at lower energies of gamma and X-rays
(on the order of keV). All the energy of the photon is transferred to the electron in
the electron shell, part of this energy is used to release the electron (output work)
and the rest is the kinetic energy of the generated photoelectron (Fig. 14.12). The
resulting photoelectron can ionize its surroundings. This phenomenon is typical for
X-rays or low-energy gamma rays.

secondary electron

primary photon

Fig. 14.12: The scheme of photoelectric effect.

• Compton scattering – occurs when gamma and X-ray photons (with higher
energy, hundreds of keV) interact with the matter. During this interaction, the
photon transfers only part of its energy to the electron of the absorbent material.
The electron gains considerable kinetic energy during the interaction and leaves the
atom (Fig. 14.13). The secondary photon (with lower energy than the primary
photon and longer wavelength) is scattered.

secondary electron

primary photon

secondary photon

Fig. 14.13: The scheme of Compton scattering.

• formation of electron-positron pairs – this phenomenon occurs at high photon

energies (of the order of MeV). The energy of a photon is transformed into the mass
and kinetic energy of an electron and a positron. The photon gets close to the
nucleus (that takes over part of the momentum) and the gamma photon is converted
217
 into an electron and a positron. The resulting secondary electrons e- and positrons
e+ (representing secondary beta particles) ionize and excite the medium (Fig.
14.14). Since the lifetime of the positron is very short 10-7 s, positron e+ reduces its
energy, it combines with any electron e-. This process is called annihilation. Two
new photons are formed (each with an energy of 0.511 MeV - which corresponds
to the energy equivalent of the resting mass of an electron or positron). The
phenomenon of electron and positron annihilation is used in nuclear medicine, the
diagnostic method is known as positron emission tomography.

Fig. 14.14: The scheme of the formation of electron – positron pairs. Annihilation of positron (right side of
the picture).

These processes of gamma radiation interaction with matter can take place
simultaneously and are governed by the exponential law (Lambert's law), which describes
the absorption of any electromagnetic field in the environment.

𝐼 = 𝐼0 𝑒 −𝜇𝑑

where (I) is the intensity of radiation (electromagnetic field) at depth (d) in the material (after
passing through the material), (I0) is the intensity of the radiation (e.g. number of photons)
coming into the absorbing material, (d) is the thickness of the absorbent material and (μ) is
the linear absorption coefficient.
The interaction of gamma radiation and the absorber is characterized by a half-
thickness d½ (half-layer). It is the thickness of the absorbent material that halves the intensity
of the incident radiation. Its value depends on the type of radiation and the radiation energy
(Fig. 14.15).
The linear absorption coefficient (μ) characterizes the attenuation of the gamma
radiation as it passes through the substance. It is actually a macroscopically effective cross-
section for beam particle loss. Its unit is m-1 (cm-1).

218
 The relation between half-thickness and the linear absorption coefficient is: d½ = ln2 /
μ. Then linear absorption coefficient: μ = ln2 / d½.

Fig. 14.15: Penetrative power of various types of ionizing radiation.

Measurement of gamma-rays absorption

Task:
1. Measure the intensity of the incident gamma radiation expressed by the number of
pulses within 10 s and its intensity after passing through different absorber thicknesses.
2. Determine the half-layer (d1/2) and calculate the linear absorption coefficient (µ).

Procedure:
1. Switch the source on, set the time preset to 10 s, and start the measurement by pushing
the “NULA” and “START” buttons, after 10 s read the number of impulses. Repeat
the measurement three times.
2. Remove the cesium emitter and plates from the lead container. Measure the
background value at the empty container.
3. Using tweezers put the emitter (137Cs) into the lead container of the detector and
measure the radiation (N0p) three times.
4. Stepwise add individual plates of absorption material between the emitter and the
scintillation detector, measure the number of impulses in 10 s, and repeat each
measurement 3 times.
5. Calculate the arithmetic means, construct a graph (the dependence of radiation
intensity on the thickness of the absorption material), determine the half-layer (d1/2),
and calculate the linear absorption coefficient.
219
Table of recorded values: N
[imp/time]
Impulses / 10 s
1 2 3 mean
Nbackground
N0p
N1p
N2p
N3p
N4p
0 d [mm]

Conclusion:

Biological effects of ionizing radiation

All types of ionizing radiation result in biological effects in the irradiated organism.
Ionization and excitation disrupt the chemical bonds between atoms and molecules, resulting
in secondary electrons and a series of free radicals that induce changes in cells - damage or
death. The effectiveness of the radiation depends on several properties and factors
throughout the irradiation process mainly the energy absorbed.
Ionizing radiation causes changes in the DNA of chromosomes. There may be a so-
called one-chain break or complete break in DNA that can no longer be repaired. Most minor
damage can be repaired by the body through repair processes.

The effects of ionizing radiation in terms of time are divided into:

• early – acute radiation sickness, acute local changes, fatal damage,
• late – local, general.

220
In terms of the probability of side effects, these are divided into:
• deterministic (probable),
• stochastic (random).

Deterministic effects
These effects occur only if higher than a certain threshold dose for the occurrence of
changes and damage in the body. The effect (damage) increases with increasing dose. In
radiotherapy, critical organs are covered by e.g. lead covering blocks in order to keep their
exposure below the threshold. A typical example is the covering of the spinal cord during
abdominal irradiation.

Stochastic effects
The stochastic effects are threshold-free (at least there is no clear level) and the damage
to the body increases with increasing dose. However, even small doses of radiation can cause
irreversible changes. So, the effects on the body are accidental, individual, and
unpredictable. The severity and course of the damage or disease do not depend (weakly
depends) on the absorbed dose (Fig. 14.16).

Fig. 14.16: Relationship between radiation dose and effect in deterministic (left) and stochastic radiation
effects (right).

Fig. 14.17: Tissue sensitivity to ionizing radiation.

In terms of the sensitivity of tissues and organs to ionizing radiation, we can rank
the organs in ascending order from the least to the most sensitive (Fig. 14.17).

221
Protection against ionizing radiation

Irradiation is the exposure of an organism to ionizing radiation. In clinical practice,

people receive medical exposures, but one can be irradiated (even significantly) as a result
of a radiation accident. Medical irradiation is the exposure of an organism to an artificial
source of radiation based on a medical indication. The physician should carefully consider
the indication for ionizing radiation examination (or therapy).
Radiation protection is the protection of people and the environment from
ionizing radiation and its effects. Law No. 87/2018 Coll. on radiation protection sets
exposure limits. Irradiation limits represent the upper level of exposure to the population,
students, and workers with ionizing radiation.

Quantities characterizing the source of ionizing radiation:

• activity A – the number of disintegrations in the sample per second. It is a basic
quantity that characterizes the source of ionizing radiation. The unit of activity is
Becquerel (Bq) – 1 Bq represents one radioactive transformation per second.
Previously, a Currie (Ci) unit was used, which corresponded to an activity of 1g
222
Ra. A conversion relationship applies between Bq and Ci: 1Ci = 3.7.1010 Bq.
Quantities characterizing spontaneous decay of atomic nuclei:
• law of radioactive decay
𝑁 = 𝑁0 𝑒 −𝜆𝑡
where (N0) is the number of radioactive nuclei at time t = 0 s, (N) is the number of
undecayed (still unchanged) nuclei at time t, and (λ) is the transformation constant
(decay constant) (Fig. 14.18).

Fig. 14.18: The dependence of the number of non-disintegrated nuclei N on time t.

• decay (disintegration) constant λ – characterizes the decay rate. The unit is the
reciprocal second = s-1. It is a measure of the activity of a given radioactive element.
This quantity is not illustrative, and therefore the concept of half-life is introduced
in practice.
222
 • physical half-life – it is a time during which one-half of radioactive atoms decay
(the number of atoms is taken at the time t0 = 0 and only one-half of original
nuclei are left after the period of half-life). It is one of the basic characteristics of
a radioactive element. It is measured in time units, e.g., seconds, minutes, days,
ln2
and years. The relation between the decay constant and the half-life: 𝑇1⁄ = .
2 𝜆

Quantities characterizing the interaction of ionizing radiation with matter:

• absorbed dose (D) – the energy absorbed in 1 kg of substance. It is a basic quantity
characterizing the interaction of radiation with matter. The unit of absorbed dose is
Gray (Gy), which is 1 Joule of absorbed energy in 1 kg of substance. The older unit
of absorbed dose was Rad, where 1 Rad = 102 Gy.

Quantities used to measure irradiation:

• dose equivalent (H) – characterizes the biological activity of different types of
radiation and is defined as the product of the absorbed dose (D) in the tissue and
the radiation quality factor (Q). The unit of dose equivalent is Sievert (Sv).
𝐻 = 𝐷. 𝑄
• radiation quality factor (Q) – expresses the biological efficiency of different types
of radiation. It has different values for different types of radiation. e.g., for photon
X-ray, gamma radiation, beta Q = 1, for neutrons and protons Q = 10, for alpha
radiation Q = 20.
• radiation weighting factor (wR) – serves similarly to the radiation quality factor to
express the different biological efficiencies of different types of radiation. The
radiation weighting factor relates in particular to the late effects of radiation, e.g.,
wR = 1 for photons, electrons and muons of all energies, wR = 5 - 20 for neutrons,
wR = 20 for alpha particles and heavy nuclei.
• effective dose (E) – expresses the different radiosensitivity of different organs and
is defined as the sum of the weighted mean values of equivalent doses as follows
𝐸 = ∑𝑇 𝑤𝑇 𝐻𝑇
where (HT) is the equivalent dose in tissue or organ and (wT) is the corresponding
tissue weighting factor.

Irradiation limits in the calendar year, effective dose:

• population – 1 mSv / year
• employees – 20 mSv / year
• students (16 – 18 years) – 6 mSv / year.

Irradiation limits in the calendar year, effective dose, and equivalent dose:
• eye lens – population (15 mSv), workers (20 mSv),
• leather – population (50 mSv), workers (500 mSv),
• limbs – population (not specified), workers (500 mSv).

These limit values are determined by the Slovak Act No. 87/2018.
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15 15 Biocybernetics
The term cybernetics originates from Ancient Greek (kybernētēs, meaning steersman)
aroused as an independent scientific discipline after World War II. The man who first used
this term was an American mathematician Norbert Wiener (1894-1964). Cybernetics, one of
the fastest growing fields of science, also affects the development of all other sciences and
has an extremely close connection with biology and medicine. It deals with the problems of
information transmission and control in highly organized systems (e.g., living organisms,
computers, factories, etc.) and studies the common structural and functional principles in the
different systems. Generally speaking, biocybernetics can be described as a systematic
application of theoretical knowledge from mathematics, physics, and the technical sciences
to study biological systems.

Information and its transmission and processing

The term information refers to the most common message or data about a process that
has occurred in the system or its surroundings. Information relates to the relationships or
interactions of the systems or the elements in a system. It is a sequence of symbols (e.g.,
letters, words, pictures etc.) that express the state and behavior of a subject.
The basic unit of the information is a binary digit and most commonly is used
contraction 1 bit. A bit can store only one of two values: 0 or 1, which may correspond to
the electric off or on values. Since the bit is a very small unit, in practice a larger memory
unit, namely byte, is usually used. The byte (B) represents 8 bits (1B = 8b). However, the
byte is still small unit for today’s use, thus kilobytes (kB), megabytes (MB), gigabytes (GB),
and terabytes (TB) are used more often. Larger units are not commonly used yet, as terabytes
are sufficient nowadays.

Types of information

• analog information –information is represented by continuously variable physical

quantities in time (Fig. 15.1). Through the senses, people are able to receive only
analog information (heat, light, pressure).

Fig. 15.1: An example of an analog signal. It varies continuously in both amplitude and frequency and the
signal can take one of many different values.

224
 • digital information –information is represented by a sequence of discrete values
(Fig. 15.2). A digital signal can only take one value from a finite set of possible
values at a given time. All computers (including medical devices) work with the
information in a digital form. Digital information is processed, stored, and spreadable
well.

Fig. 15.2: The digital signal can only take on two values, either a 1 or a 0 (sometimes called 'on' or
'off').

The information forms the basis of communication between the systems, where one
system sends a message and the other receives it. The simplest information system consists
of 3 basic parts: transmitter, channel, and receiver (Fig. 15.3).

transformation
and coding decoding
information channel user
transmitter receiver
source

noise

Fig. 15.3: Block diagram of a communication system.

The information comes from the information source to the transmitting unit. A
transmitter transforms the information into a signal suitable for further transmission. In most
cases, the information (message) cannot be spread in its original form, and thus, coding is
necessary. A code is a form of a rule for converting a piece of information into another form
or representation (for example information about somebody’s name is coded by a correct
sequence of letters and word, phrase, or gesture, e.g., Michael, not Melihac). Coding is a
transcript where a certain character or letter is assigned to the information (changing
information from one form to another using an algorithm). After successful coding, the
signals are transmitted to the receiver through a channel. The channel represents the
environment (electric conductor, nerve fiber, cytoplasm, etc.) which provides the signal
transmission. In addition to signals transmitting information, also signals from the

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environment itself may also enter the channel. These signals comprise noise that can distort
the original meaning of the information, thereby disturbing its correct interpretation.
Information processing, in which signals are transformed into a directly understandable
form, takes place in the receiver. This last process is known as decoding (reverse coding
process).

Of note: Such an example could be the coding of stimulus intensity by receptors.

Increased stimulus intensity (e.g., pressure) causes an increased frequency of action
potentials in the afferent nerve fiber. However, the amplitude of the action potential does
not change. Thus, the information (action potential) is coded by a frequency, not an
amplitude (Fig. 15.4).

Fig. 15.4: Frequency pressure coding.

Biosignals

Any living organism or biological system constantly communicates (interacts) with

the surrounding environment and with other systems through information. The organism is
influenced by the surrounding environment, and thus changes its internal state (it processes
information, and adapts to the surrounding environment). A signal is known as the
information carrier. The physical carrier of the information about the activity of the
biological system and its relations with the environment is called the biosignal.

Types of biosignals

• biological signals created by vital manifestation – signals are generated by the vital
manifestations of the organism without external intervention. They reflect the
internal state of the system (manifestation of the activity of the organ such as the
heart, brain, and activity of neurons). The output signal is for example blood pressure,
respiratory rate, the blood concentration of O2, CO2, temperature, electrocardiogram
(ECG), electromyogram (EMG), electroneurogram (ENG), electroencephalogram
(EEG), and pH.
• biological signals created by stimuli from the external environment – the signals
are created by stimulation of the living system (human body). The biological system
is stimulated to obtain information about the structure, function and condition of the
system. The final biosignal results from the interaction of an external signal

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 (stimulation of the human body by X-ray or by magnetic field etc.) with the system,
e.g., SONO, MRI, CT, PET, skiagraphy, sciascopy.

Biosignals according to their physical entity

• electric biosignals – results of the electric events occurring in the plasma cell
membranes and tissues, e.g. ECG, EEG, EMG, ENG.
• non-electric biosignals – results of mechanical events in the biological system such
as blood pressure, blood flow, air flow, deflection, or movement. Non-electric signals
must be transformed into electric signals before processing (filtration,
amplification). During the transformation and signal processing, it is necessary to
preserve the information contained in it.

Acquisition and recording of the electric signals

The advantage of an electric signal is that it can be easily transmitted to a remote

monitoring point, amplified if necessary, and then displayed in a way that offers maximum
comfort for reading or measurement. The electrodes are commonly used to obtain an electric
signal.

Electric signals (Table 17.1):

• are created as a result of the electric events on the cell membranes (e.g. action
potentials),
• in living organism transmit coded information from the receptors to the brain and
from the brain to muscles, glands, organs,
• are frequently frequency coded (modulated); in living organisms the stronger the
stimulus, the higher the rate (frequency) of the action potentials from the receptors,
• are transmitted to the body surface by conductive body environment (and usually
detectable on it),
• are usually recorded by non-invasive methods,
• are easily processed by electronic devices,
• are very weak (amplitude is usually very small) so amplitude amplification is
necessary.

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 Table 17.1: Examples of the most common recorded electrical signals in medicine.

Signal Frequency band

Name of the method Amplitude [mV]
(abbreviation) [Hz]
ECG Electrocardiography 0.5 – 5.0 0.01 - 250
Electromyography
EMG 0.1 – 10.0 0.01 – 10 000
(surface electrode)
Electromyography
EMG 0.05 – 5.0 0.01 – 10 000
(needle electrode)
EEG Electroencephalography 0.01– 50.0 0.01 – 100

ENG Electroneurography 0.05 – 10.0 0.01 – 1000

The electrodes recording of the electric signals according to their physical entity:
• polarizable electrodes – no charge passes through the electrodes when current is
applied, they have a variable electrode potential. The current does not flow, but
rather changes the concentration of the ions at the interface. They are usually made
of noble metals such as gold, and platinum. They behave like a capacitor.
• non-polarizable electrodes – charge freely passes the interface when current is
applied, its potential does not change from its equilibrium potential. Non-
polarizable electrodes are used in surveys to eliminate the polarization effect that
occurs when using metal (stainless steel) electrodes, e.g., silver-chloride electrode
(Ag-AgCl). They behave like a resistor.

Of note: It is not possible to produce a perfectly polarizable or non-polarizable

electrode, the real electrode will always only approximate one of the types.

Electrodes for the recording of the electric signals according to their type:
• macroelectrodes – surface electrode, needle electrode
• microelectrodes

The surface electrode measures the potential available at the surface of the skin. It
detects the signal from the heart, the brain and the nerves. Larger surface electrodes record
the ECG signals. Smaller surface electrodes record the EMG and EEG signals. The types of
surface electrodes are as follows:

• Metal plate electrodes – made from metals of various shapes and sizes (Fig. 15.5).
They have a smaller contact area and do not seal completely on the patient.
Electrodes are fixed on the skin using electrolyte paste to reduce transitional
resistance between the skin and the electrode. The electrode slippage and movement
are two major disadvantages of this electrode type. They are very sensitive which
leads to measurement errors.

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 a) b)

Fig. 15.5: (a) Metal-plate electrode used for application to the limbs. (b) Metal disk electrode applied by
surgical tape.

• Suction cup electrodes – they have a squeezable rubber bulb, a metal contact
surface, and contact cables (Fig. 15.6). They have a good contact area; no electrolyte
paste application is necessary.

Fig. 15.6: A metallic suction electrode.

• Adhesive type electrodes – they have an adhesive layer (Fig. 15.7) with a
lightweight metallic screen and a pad at the back for placing electrode paste. This
adhesive backing holds the electrode in place and tightens it. It also helps to avoid
the evaporation of electrolytes present in the electrode paste.

Fig. 15.7: An adhesive type electrode.

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 • Floating electrodes – they do not touch the human body directly. They contact the
skin via electrolytic paste or conductive jelly. The principle of this electrode is to
eliminate motion artifacts by avoiding the direct contact of the metal with the skin.
The electrodes are attached to the skin by two adhesive rings, which adhere to both
the plastic surface of the electrode and the skin (Fig. 15.8).

Fig. 15.8: Example of floating electrode. (a) schematic view, (b) cross-section view.

Needle electrodes or intramuscular wire electrodes have advantage over surface

electrodes that they are able to record activity from the individual motor units. Recording
and evaluation of the action potentials are more precise. Needle electrodes are used for the
recording of the action potentials from the muscles or together with the hypodermic
electrodes for long-term recording of the heart or the brain potentials. The electrode itself is
a noble metal conductor which is insulated by a hypodermic needle (Fig. 15.9 A). The fine
wire electrode is inserted into the muscle of interest. The needle or steel cannula is removed
and the electrode wires are attached to the steel spring adapters to minimize movement
artifacts (Fig. 15.9 B). The disadvantage of using these electrodes is the discomfort for
patients or test subjects.

Fig. 15.9 A: Bipolar wire electrode with hook shaped tip.

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 Fig. 15.9 B: Bipolar wire electrode inserted in the muscle.

Microelectrodes are used to study the electrophysiology of the excitable cells by

measuring the potential differences across the cell membrane. A microelectrode must be
very small and strong (rigid) to penetrate the cell membrane without damaging the cell. They
are usually made of a glass capillary filled with the electrolyte solution or of a thin
borosilicate glass with carbon fiber (Fig. 15.10). They have a very thin tip diameter between
1-15 µm and high input impedance.

a) b)

Fig. 15.10: Microelectrodes: (a) glass capillary can be filled with NaCl electrolyte solution for extracellular
recording or KCl solution for intracellular recording (b) carbon fiber microelectrode.

In general, biologic/bioelectric signals have a low amplitude. Because the amplitude

is so low that it cannot be processed directly. The amplifiers are used to increase the
amplitude of the biosignals. The outputs from the amplifiers are analyzed while they appear
as bioelectric waveforms (e.g., ECG, EMG, or others). Such amplifiers are defined as Bio
Amplifiers or Biomedical Amplifiers. The bio amplifier is a functional unit (device)
consisting of an amplifier and another electronic element. The signal connected to its input
appears at the output in an enhanced (amplified) form (Fig. 15.11). The amplifier must
replicate the original signal without distortion so that none of the information
contained in the original signal is lost!

input output

Fig. 15.11: Amplifier and its block scheme (output signal is amplified / enhanced).

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 The basic characteristics of the amplifiers are input and output parameters (e.g.,
voltage, resistance, amplification, transmitted band, etc.). Any biological amplifier should
have a high input impedance, usually between 2 MΩ and 10 MΩ depending on the
application. A higher impedance value reduces distortion of the signal. When electrodes
record biopotentials from the human body, the input circuit should be protected by isolation
and protection circuits, to prevent the patients from electric shocks. There are several types
of bio amplifier: differential amplifier, operational amplifier, isolation amplifier.

The principles of transducers for the recording of non-electric signals

A number of physiological measurements are made by using sensors that convert

non-electric signals into electric signals. Such devices are often called transducers.
Depending on their physical principles, they can be divided into electromechanic,
electroacoustic, electrochemical, thermoelectric, and radioacoustic.

• Electromechanic transducer – a device for converting mechanic motion

(vibrations) into variations of an electric current or voltage (electric signals) and vice
versa. Typical examples are air flow sensors (spirometry) or pressure sensors (blood
pressure monitoring – arterial line, Fig. 15.12). The arterial line (invasive blood
pressure measurement) is most commonly used in intensive care medicine and during
anesthesia to monitor blood pressure directly in real-time and with high accuracy and
to obtain samples for arterial blood gases analysis.

Fig. 15.12: Schematic representation of the arterial line (direct or invasive blood pressure measurement).
The catheter with saline in the arterial set transmits the pressure changes to the diaphragm (membrane) in
the transducer (right upper corner). The transducer “reads” the changing pressure, and changes in into an
electric signal that goes up and down as the pressure does, which is displayed as an arterial waveform.
Disposable blood pressure transducer (right bottom corner).

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 • Electroacoustic transducer – a device that converts acoustic energy (sound
vibrations: oscillations in air pressure) into electric energy (voltage variations). A
typical example is a microphone, an ultrasound probe (see chapter 9), or piezoelectric
crystals (Fig. 15.13).

Fig. 15.13: Left: An example of an electroacoustic transducer (inductive transducer - microphone) - self-
inductance of a single coil is changed by a quantity (sound waves) that is to be measured. Right: the
electric charge is accumulated in certain solid materials (such as crystals, certain ceramics, and biological
matter such as bone, DNA and various proteins) in response to applied mechanic stress.

• Electrochemical transducer (sensor) – converts the information associated with the

electrochemical reactions (the reaction between an electrode and an analyte) into an
applicable qualitative or quantitative signal. A typical example is a pH probe.
Electrochemical transducers are the most commonly used transducers in the
construction of innovative sensors including biosensors (Fig. 15.14).

Fig. 15.14: The structure of a biosensor.

• Thermoelectric transducer – a device that transforms a temperature change into an

electric current. Changes result from temperature differences at the junction of two
different materials. The thermal electric transducer is more known as
a thermocouple. A device that transforms temperature into an electric resistance is
called a resistance thermometer.

• Radioacustic transducer – a device that transforms incident ionizing radiation to an

electric impulse e.g. Geiger–Müller tube or converts electromagnetic transmissions
to electric signals, e.g. radio receiver.

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Monitoring and telemetry

Medical monitoring is the monitoring of a disease, patient condition, or one or more

medical parameters over time. Numerical values are usually obtained from a display device
such as a medical monitor. There are several types of digital displays. Current biomedical
meters use light-emitting diodes (LEDs) and liquid crystal diodes (LCDs). Using
semiconductor digital circuits, the voltage can be converted to a set of “small” voltages that
are applied to the appropriate electrodes in an LED or LCD array to create visible symbols
(e.g., letters) or a waveform corresponding to the voltage. Once the electric signal is
converted to the digital form, it is easier to add logic or computational circuits to perform
other useful functions (processing). For example, when a temperature probe is inserted into
the mouth, the sequential temperature is displayed. The associated circuits sense when the
probe temperature has reached its equilibrium value, based on the stable temperature changes
(that no longer shift). The thermometer then produces a signal indicating that the temperature
has reached a stable value and can be recorded.
A patient monitoring system (e.g. electronic vital sign monitor, Fig. 15.15 LEFT) is
the set of systems and/or processes that enable healthcare providers to monitor a patient's
health status. In general, a patient monitoring device typically contains a sensor for recording
important patient information (e.g., heart rate) and connections (e.g., PCBs, connectors,
wiring, etc.) that can transmit the information to the display device, e.g. a pulse oximeter
measures and transmits patient’s pulse to the main monitoring device.
The basic monitors show the patient's heart rate, blood pressure, and body
temperature. More advanced models also show how much oxygen a patient has in the
blood or how fast a patient breathes (Fig. 15.15 RIGHT). Some models have also the ability
to show intracranial pressure or carbon dioxide concentration in exhaled air. The monitor
will make certain sounds if any of the vital signs fall below safe levels.

Fig. 15.15: LEFT: Patient Monitoring Device, Usage: Hospitals, Clinical Use, Outpatients Centre; RIGHT:
Most often patient vital signs.

It may be necessary to process the signal at some distance from the signal source. A
well-known example is the monitoring of the vital functions of astronauts in space or sports
physiology on Earth. In this case, only wireless communication is possible, because the wires
would hinder the activities to be observed. Transmitting data from a signal source or a
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monitor to a distant monitoring station is known as telemetry or biotelemetry. The hospital
telemetry unit (Fig. 15.16) can be an essential aspect of medical care. Remote monitoring of
cardiac patients using sophisticated telemetry equipment after surgeries, procedures, and
other cardiac treatments or interventions are examples of what a telemetry unit is for. When
patients enter a hospital's telemetry unit, they are usually cared for by a telemetry nurse.

Fig. 15.16: Left: Telemetry unit; Right: Hospital patent telemetry monitoring system.

Systems – the terminology and their structure

The term system refers to a group of interacting or interrelated elements that are
organized in a unified entity. A system is surrounded and influenced by its environment and
has defined boundaries, structure, and function. There are several types of systems. They are
studied by system theory and systems science.

• Technical systems – are artificially created by humans (Fig. 15.17).

Fig. 15.17: Examples of technic systems; LEFT: Engine; MIDDLE: An electrical circuit; RIGHT: An
algorithm.

• Biological systems – represent a complex network that connects several biologically

relevant entities. At the microscopic level, examples of the biological systems are
cells and organelles, at the macroscopic level, examples are tissues and organs.
Specific (multilevel) systems are represented by the circulatory system, respiratory
system, nervous system, and several regulatory pathways such as the endocrine
system or metabolism. (Fig. 15.18).

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 Fig. 15.18: Examples of biological systems; LEFT: Cell; MIDDLE: Brain; RIGHT: Metabolism.

According to the relationship of the systems to the external environment, the systems
are divided into:
• Isolated system – does not interact with the environment. The system exchanges
neither matter nor energy (they cannot enter or exit the system). An example of an
isolated system is a steel thermos flask (Fig. 15.19) keeping the mass (tea) hot or cold
(constant energy) for a long time. However, a truly isolated system cannot exist in
nature. Even the flask will eventually cease to function as an isolated system - the tea
will be cooled and evaporated eventually (after a long time).

Fig. 15.19: Examples of isolated system – the steel thermos flask, neither matter nor energy can enter or
exit the system.

• Closed system – certain interactions with the surrounding environment are possible.
A closed system exchanges energy with the environment, but not matter. An example
of a closed system can be a cup of tea with a lid (Fig. 15.20) - a cup with a lid keeps
the matter (tea) for a long time, but it is warm (energy) for a short time.

Fig. 15.20: Examples of closed system – a cup of tea with a lid, energy can enter or exit to system.

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 Open system – all interactions with the surrounding environment are possible. Open
system exchanges both energy and matter with its surroundings. An example of
anopen system can be a living cell (15.21).

Fig. 15.21: Examples of open system – a living cell, both energy and matter exchange are possible.

According to the behavior of the system over time, the systems can be divided into:
• Static system – there is no change (no behavior of a system) in time. The elements’
interactions (relations) are stable in time.

• Dynamic system – system behavior is changing over time. The elements of a system
interact with each other differently in time. The interaction may be of a substantial
nature (e.g., organelles in a cell).

The dynamic systems properties

A living cell is an example of an open dynamic system. The cell interacts with the
environment (exchanges matter and energy) and the cell changes over time (grows and ages,
etc.). The basic feature of a dynamic system is its multiple and variable interactions with the
environment. We can act on the open system through input quantities (inputs) and the system
can act on its surroundings through output quantities (outputs) (Fig. 15.22).

Fig. 15.22: Open system diagram with its input and output flows.

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 While the input variables are independent, the output variables depend on both the
input variables and the internal properties of the system. Input and output quantities of a
biological system can be of a material, energy, or information nature. An example of a single
input system is the receptor, where the input variable is e.g. pressure (for mechanoreceptor)
and the output is the frequency of the action potentials in the afferent fiber. Biological
systems usually have multiple inputs and outputs, e.g. retina, where the rods and cones are
the input receptors and the optic nerve fibers carry the output quantities.

According to the behavior of the system, the systems can be divided into:
• Stochastic systems – the behavior of the system is random, and unpredictable,
caused mainly by random factors, e.g. nongenetic cell variability, and diseases
associated with ionizing radiation exposure.
• Deterministic systems – the behavior of the system is predictable, and clearly
determined by its status and stimuli, e.g. genetic cell variability, viral disease,
diabetes.

Stochastic processes (e.g., non-genetic variability) form the basis for the rapid
adaptation of the organism to a changing environment. While deterministic processes (e.g.
genetic variability) are the basis of adaptation in the long-term perspective. A living
organism is a combination of deterministic and stochastics processes, which represent an
elegant way to increase biological complexity with a limited number of genes. A living cell
can be defined as an open dynamic system in which a combination of deterministic and
stochastic processes takes place.

A relationship (coupling) is created between the input and output of two or more
subsystems. The coupling can be serial, parallel, or feedback (Fig. 15.23).

a)

b)

c)
S1 S2

Fig. 15.23: Basic types of bonds in dynamic systems. a) serial, b) parallel, c) feedback.

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 Feedback is especially important in biophysics. The term feedback refers to the
effect of the system output quantity on its own input. The feedback can be positive, negative
or the system without feedback.

• No feedback (Fig. 15.24) – it is a simple system control, where the output depends
only on the input. An example of non-feedback control is hair growth or single action
potential.

input output
System

Fig. 15.24: System control diagram without feedback.

• Positive feedback (Fig. 15.25) – positive feedback control stimulates physiological

processes. System output affects system input in terms of amplification (a stronger
output amplifies the input). Positive feedback is cumulative. It is used where a small
stimulus needs to lead to a strong response e.g. growth and development of
organisms, stress response, etc.

Of note: Platelets attach to the affected area and secrete chemicals that attract more
platelets, which excrete more chemicals, and the cycle is repeated until the hemorrhage
stops. The positive feedback mechanism also applies during the birth of a child (labor).

+ +++
System (+)
+

Fig. 15.25: Positive feedback system control diagram.

• Negative feedback (Fig. 15.26) – negative feedback control is a process that

suppresses (regulates, downregulates, keeps in the range) physiological processes.
System output affects system input in terms of input attenuation (a stronger output
attenuates the input). Negative feedback is compensatory. It is a process that arises
when the system needs to slow down (attenuates) or completely terminate a
physiological process. Negative feedback control keeps stability and homeostasis in
the system.

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 +++ +
System (-)
-

Fig. 15.26: Negative feedback system control diagram.

Of note: Almost all the control mechanisms in the human body that provide
homeostasis are based on negative feedback, e.g. when blood glucose levels begin to rise,
the pancreatic beta cells begin to secrete insulin, which lowers blood glucose levels. The
cycle is repeated until the blood glucose level is normal.

Dynamic systems have the ability to convert the values of the input variables to the
values of output variables. The basic properties of the dynamic systems include signal
amplification or attenuation, signal delay, semipermeability (signal filtering),
implementation of certain logic functions, etc. All these properties are present in the
biological system, e.g. amplification is applied to sensory perception, attenuation leads to
system homeostasis, etc. The relationships between input and output quantities of a given
dynamic system can change a lot. Dynamic systems are capable of both adaptation and
learning. In the case of the biological systems, these relationships are affected by growth,
development, aging, but also disease.

Of note: Sensory receptors respond to threshold stimuli (stimuli of a very low energy
level). The input signal is transformed by the receptor into a "standard" energy level of the
nervous system signal, which is necessary for the correct transmission and resolution of
signal features.

The basics of organ control and regulation

Control and regulation are processes that effectively maintain the required state in
dynamic systems. According to the complexity of the process, the systems can be divided
into controlled (without feedback) and regulated (with feedback). The term control refers
to the process, which occurs when the control part sends the information to the part which is
controlled (Fig. 15.27).

controlling control controlled

part part

Fig. 15.27: System control scheme.

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The control modes in the living organisms can be divided into:
• Direct control – is the easiest form of system control. It is a straight relationship
between the control part and the controlled part (Fig. 15.27). The behavior of the
controlled part is deterministic and dependent on the control part.

• Autonomous control system – system commands represent the implementation

process in the system. The system has several degrees of autonomy. The controller
is hierarchical, with an execution level (the lowest level), coordination level (middle
level), and management and organization level (highest level).

A process that minimizes the difference between the actual values of the controlled
variables and their set (required) points is regulation, e.g. cells (organ), which regulate some
quantity (blood glucose level, blood pH, body temperature, etc.) represent the homeostatic
regulatory system. Regulation is a cycle of events in which the status of the monitored value
(e.g. temperature) is constantly monitored and the information about it is sent to the control
center. It receives the information about the system state from the receptors that detect
values and send information to the control center. The control center determines the “ideal”
value at which the state of the system should be maintained. The control center reacts through
effectors guided by the control center. Effectors execute the response (Fig. 15.28).

Receptor Effector
Stimulus

Control center
(CNS)

Fig. 15.28: Control and regulation scheme; the red arrow represents the input / output information; the blue
arrow represents positive / negative feedback.

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16 16 Quantities and units

Scalar quantities are fully determined only by the numerical magnitude (of course
by a unit of the particular quantity, e.g. length, volume, mass, time, etc.). Vector quantities
are determined by the magnitude and unit but also their direction and orientation (e.g., force,
acceleration, etc.).
Each quantity is determined quantitatively (the magnitude) and qualitatively (which
quantity is expressed). The unit of a physical quantity is a standard quantity chosen as a basis
for comparing with it.
The unit may be chosen arbitrarily for each quantity, however, that would lead to unit
system congestion. That is why the set of mutually independent units was chosen – basic
units. All the other units may be derived from them – derived units.

The international system of units – SI (Système International) was codified in 1960 at

XI. General Conference on Weights and Measures as a universal system of measures. The
bases of this system are:
a) 7 base units,
b) 2 dimensionless units,
c) derived units,
d) multiples and portions of the units.

SI units

The international system of units – SI (Système International) was codified in 1960

at XI. General Conference on Weights and Measures as a universal system of measures. SI
(basic) units involve the most commonly used physical units:

1. meter [m] – unit of length. It is defined as the length of the path traveled by light in a
vacuum during a 1/299,792,458 of a second.
2. kilogram [kg] – unit of mass. Since 2019, a kilogram is defined by taking the
numerical value of the Planck constant h to be 6.62607015×10−34 when expressed in
the unit J⋅s, which equals kg⋅m2⋅s−1. The exact determination of kg triggered
redefining other basic units as well.
3. second [s] – unit of time. Represents the duration of 9 192 631 770 radiation periods
corresponding to the transition between the two hyperfine levels of the ground state
of the cesium 133Cs atom.
4. ampere [A] – unit of electric current. Since 2019, one ampere equals the flow of
6.2415093 × 1018 elementary charges (such as electrons, the definition of Coulomb),
through the conductor point in 1 second.
5. kelvin [K] – unit of thermodynamic temperature. Since 2019, kelvin is defined by the
numerical value of the Boltzmann constant k fixed to 1.380649×10−23 J⋅K−1. A

242
 change in one kelvin of the thermodynamic temperature T results in a change of
thermal energy kT by 1.380649×10−23 J.
6. mole [mol] – unit of amount of substance. Since 2019, one mole of a substance was
redefined as containing exactly 6.02214076×1023 elementary entities of the
substance, wherein this number represents a fixed numerical value of the Avogadro's
constant - the amount of substance of a system is a measure of the number of
specified elementary entities, which entities may be atoms, molecules, ions,
electrons, other particles or specified groups of particles.
7. candela [cd] – unit of luminous intensity. Represents intensity, in a given direction,
of a source that emits monochromatic radiation of frequency 540×1012 hertz and has
a radiant intensity in that direction of 1/683 watt per steradian.

Basic SI units

Unit Dimension
Basic unit Quantity name
symbol symbol
meter m length l
kilogram kg weight m
second s time t
ampere A el. current I
kelvin K thermodynamic temperature T
mole mol amount of substance n
candela cd luminous intensity I

Derived SI units

SI-derived units are defined in terms of the seven SI base units via a system of
equations defining particular derived quantities. The derived units are coherent with the basic
units. Derived units are formed by a combination of basic units, due to their length and
complexity, some of them are renamed, for example becquerel, coulomb, farad, gray, henry,
hertz, joule, lumen, lux, newton, ohm, pascal, siemens, tesla, volt, watt, weber, etc.
The following table lists the most frequently used derived units.

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 Derived units

Derived unit Expression

In terms of In terms of
Derived quantity Name Symbol other SI base SI units
units

Plane angle radian rad m . m–1 = 1

Solid angle steradian sr m2 . m–2 = 1
Frequency hertz Hz s–1
Force newton N m . kg . s–2
Pressure pascal Pa N . m–2 m–1 . kg . s–2
Energy, work, quantity of heat joule J N.m m2 . kg . s–2
Power, radiant flux watt W J . s–1 m2 . kg . s–3
Electric charge coulomb C A.s
Electric potential, potential
difference, voltage, volt V W . A–1 m2 . kg . s–3 . A–1
electromotive force
Capacitance farad F C . V–1 m–2 . kg–1 . s4 . A2
Electric resistance ohm  V . A–1 m2 . kg . s–3 . A–2
Electric conductance siemens S –1 m–2 . kg–1. s–3 . A2
Magnetic flux weber Wb V.s m2 . kg . s–2 . A–1
Magnetic flux density tesla T Wb . m–2 kg . s–2 . A–1
Inductance henry H Wb . A–1 m2 . kg . s–2 . A–2
Luminous flux lumen lm cd . sr
Illuminance lux lx lm . m–2 m–2 . cd . sr
Activity (of a radionuclide) becquerel Bq s–1
Absorbed dose, specific energy
(imparted), kerma, index of gray Gy J . kg–1 m2 . s–2
absorbed dose
Dose equivalent sievert Sv J . kg–1 m2 . s–2
Catalytic activity katal kat mol . s–1

Other units are only used in special areas.

Unit Area of
Quantity
Name Symbol Value in SI units application
The optical power of dioptria
D 1 D = 1 m-1 optics
the optical system (diopter)
The pressure of blood Mercury
mmHg 1 mmHg = 133,322 Pa healthcare
and other body fluids millimeters

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Units are defined based on SI units that are not decimal multiples of SI units.

Unit
Quantity Note
Name Symbol Value in SI units
minute min 1 min = 60 s
Time hour h 1 h = 3 600 s
day d 1 d = 86 400 s

no international Do not use

revolution symbol 1 revolution = 2 rad prefixes
Plane to create
angle (angular) degree  1 = (/180) rad multiples.
(angular) minute  1 = (/10 800) rad
(angular) second  1 = (/648 000) rad
gon or grad gon 1 gon = (/200) rad

Decimal multiples of SI units with special names.

Unit
Quantity
Name Symbol Value in SI units
Volume litre l or L 1 l = 1 dm3 = 10–3 m3
Mass tonne t 1 t = 1 Mg = 103 kg
Pressure bar bar 1 bar = 105 Pa

Other units of pressure are used in medicine.

unit 1 Pa 1 atm 1 mm Hg =1 1 mm H2O

1 Pa 1 9.87 x 10 –6
7.5torr
x 10–3 0.102
3
1 atm 101.3 x 10 1 760 10.3 x 10-3
1 mm Hg =1 torr 133 1.32 x 10–3 1 13.6
1 mm H2O 9.81 9.68 x 10–5 73.5 x 10–3 1

245
Prefixes

Prefixes are added to the unit to form multiples and portions of the units. They are
created according to the third power of ten (103) by means of standardized prefixes that are
added to the name of the unit to form a single word without any conjunctive symbol.
The prefixes do not combine! For example, a millionth of a meter is defined as a
micrometer, not a millimillimeter. Multiples of the kilogram are named as if the gram were
the base unit, so a millionth of a kilogram is a milligram, not a microkilogram. However, on
the other hand, some prefixes have special names commonly used, e.g. 1000kg is known as
a ton, not as a megagram. The prefix and the symbol are put in front of the name and symbol
of the unit (e.g., nanomole - nmol; kilometer - km; picofarad - pF; nanogram – ng, etc.).

Prefix Multiplication
Name Symbol factor
exa E 1018
peta P 1015
tera T 1012
giga G 109
mega M 106
kilo k 103
mili m 10–3
micro  10–6
nano n 10–9
pico p 10–12
femto f 10–15
atto a 10–18

In special cases, prefixes with gradation by one decimal may be used.

Prefix Multiplication
Name Symbol factor
hecto h 102
deka da 10
deci d 10–1
centi c 10–2

246
 A Annex - Tables for practical tasks
Greek alphabet

α Α alpha ι Ι iota ρ Ρ rho

β Β beta κ Κ kappa σ Σ sigma
γ Γ gamma λ Λ lambda τ Τ tau
δ Δ delta μ Μ mu υ Υ upsilon
ε Ε epsilon ν Ν nu φ,  Φ phi
ζ Ζ zêta ξ Ξ xi χ Χ chi
η Η êta ο Ο omikron ψ Ψ psi
θ,  Θ thêta π Π pi ω Ω omega

Dependence of density and viscosity of distilled water on temperature.

Temperature t Density  Viscosity  Surface tension σ

C [kg.m–3] [10–3.N.s.m–2] [N.m-1]
19 998.406 1.027 0.0729
20 998.205 1.002 0.0728
21 997.994 0.978 0.0726
22 997.772 0.955 0.0725
23 997.540 0.933 0.0723
24 997.299 0.911 0.0721
25 997.047 0.890 0.0720
26 996.786 0.871 0.0718
27 996.515 0.851 0.0717
28 996.235 0.833 0.0715
29 995.946 0.815 0.0714
30 995.649 0.798 0.0712

Values of minimum and maximum erythrocyte resistance in some blood diseases

Minimal osmotic Maximal osmotic

Disease
resistance (% NaCl) resistance (% NaCl)
Erythremia 0.40 0.28
Iron deficiency anemia 0.38 0.28
Thalassemia 0.38 0.20
Drug-induced anemia 0.50 0.40
Hepatitis C 0.34 0.22
Physiological value 0.44 0.32
Hereditarian spherocytosis 0.68 0.46
Megaloblastic anemia 0.48 0.36
247
The pressure of saturated water vapor in kPa at temperature t in °C.

t .0 .1 .2 .3 .4 .5 .6 .7 .8 .9
0 0,61 0,62 0,62 0,62 0,63 0,63 0,64 0,64 0,65 0,65
1 0,66 0,66 0,67 0,67 0,68 0,68 0,69 0,69 0,70 0,70
2 0,70 0,71 0,72 0,72 0,73 0,73 0,74 0,74 0,75 0,75
3 0,76 0,76 0,77 0,77 0,78 0,78 0,79 0,80 0,80 0,81
4 0,81 0,82 0,82 0,83 0,84 0,84 0,85 0,85 0,86 0,87
5 0,87 0,88 0,88 0,89 0,90 0,90 0,91 0,92 0,92 0,93
6 0,94 0,94 0,95 0,96 0,97 0,97 0,97 0,98 0,99 1,00
7 1,00 1,01 1,02 1,02 1,03 1,03 1,04 1,05 1,06 1,06
8 1,07 1,08 1,09 1,10 1,10 1,11 1,12 1,12 1,13 1,14
9 1,15 1,16 1,16 1,17 1,18 1,19 1,20 1,20 1,21 1,22
10 1,23 1,24 1,24 1,25 1,26 1,27 1,28 1,29 1,30 1,30
11 1,31 1,32 1,33 1,34 1,35 1,36 1,37 1,38 1,38 1,39
12 1,40 1,41 1,42 1,43 1,44 1,45 1,46 1,47 1,48 1,49
13 1,50 1,51 1,52 1,53 1,54 1,55 1,56 1,57 1,58 1,59
14 1,60 1,61 1,62 1,63 1,64 1,65 1,66 1,67 1,68 1,70
15 1,71 1,72 1,73 1,74 1,75 1,76 1,77 1,78 1,80 1,81
16 1,82 1,83 1,84 1,85 1,87 1,88 1,89 1,90 1,91 1,93
17 1,94 1,95 1,96 1,98 1,99 2,00 2,01 2,03 2,04 2,05
18 2,06 2,08 2,09 2,10 2,12 2,13 2,14 2,16 2,17 2,18
19 2,20 2,21 2,23 2,24 2,25 2,27 2,28 2,30 2,31 2,32
20 2,34 2,35 2,37 2,38 2,40 2,41 2,43 2,44 2,46 2,47
21 2,49 2,50 2,52 2,54 2,55 2,57 2,58 2,60 2,61 2,63
22 2,65 2,66 2,68 2,69 2,71 2,73 2,74 2,76 2,78 2,79
23 2,81 2,83 2,85 2,86 2,88 2,90 2,92 2,93 2,95 2,97
24 2,99 3,00 3,02 3,04 3,06 3,08 3,10 3,11 3,13 3,15
25 3,17 3,19 3,21 3,23 3,25 3,27 3,29 3,30 3,32 3,34

248
Line nomogram to determine a body surface from height and weight.

HEIGHT SURFACE WEIGHT

249
 Calibrated table of refractive indices for immersion refractometer.

section .0 .1 .2 .3 .4 .5 .6 .7 .8 .9
12 1,332040 1,332 079 1,332 118 1,332 157 1,332 196 1,332 235 1,332 274 1,332 313 1,332 352 1,332 391
13 1,332 420 1,332 458 1,332 496 1,332 534 1,332 572 1,332 610 1,332 648 1,332 686 1,332 724 1,332 762
14 1,332 810 1,332 849 1,332 888 1,332 927 1,332 966 1,333 005 1,333 044 1,333 083 1,333 122 1,333 161
15 1,333 200 1,333 239 1,333 278 1,333 317 1,333 356 1,333 395 1,333 434 1,333 473 1,333 512 1,333 551
16 1,333 590 1,333 629 1,333 668 1,333 707 1,333 746 1,333 785 1,333 824 1,333 863 1,333 902 1,333 941
17 1,333 970 1,334 008 1,334 046 1,334 084 1,334 122 1,334 160 1,334 198 1,334 236 1,334 274 1,334 312
18 1,334 360 1,334 399 1,334 438 1,334 477 1,334 516 1,334 555 1,334 594 1,334 633 1,334 672 1,334 711
19 1,334 740 1,334 778 1,334 816 1,334 854 1,334 892 1,334 930 1,334 968 1,335 006 1,335 044 1,335 082
20 1,335 130 1,335 169 1,335 208 1,335 247 1,335 286 1,335 325 1,335 364 1,335 403 1,335 442 1,335 481
21 1,335 510 1,335 549 1,335 588 1,335 627 1,335 666 1,335 705 1,335 744 1,335 783 1,335 822 1,335 861
22 1,335 890 1,335 928 1,335 966 1,336 004 1,336 042 1,336 080 1,336 118 1,336 156 1,336 194 1,336 232
23 1,336 280 1,336 319 1,336 358 1,336 397 1,336 436 1,336 475 1,336 514 1,336 553 1,336 592 1,336 631
24 1,336 660 1,336 698 1,336 736 1,336 774 1,336 812 1,336 850 1,336 888 1,336 926 1,336 964 1,337 002
25 1,337 040 1,337 078 1,337 116 1,337 154 1,337 192 1,337 230 1,337 268 1,337 306 1,337 344 1,337 382
26 1,337 420 1,337 458 1,337 496 1,337 534 1,337 572 1,337 610 1,337 648 1,337 686 1,337 724 1,337 762
27 1,337 810 1,337 849 1,337 888 1,337 927 1,337 966 1,338 005 1,338 044 1,338 083 1,338 122 1,338 161
28 1,338 190 1,338 228 1,338 266 1,338 304 1,338 342 1,338 380 1,338 418 1,338 456 1,338 494 1,338 532
29 1,338 570 1,338 608 1,338 646 1,338 684 1,338 722 1,338 760 1,338 798 1,338 836 1,338 874 1,338 912
30 1,338 950 1,338 988 1,339 026 1,339 064 1,339 102 1,339 140 1,339 178 1,339 216 1,339 254 1,339 292
31 1,339 330 1,339 368 1,339 406 1,339 444 1,339 482 1,339 520 1,339 558 1,339 596 1,339 634 1,339 672
32 1,339 710 1,339 748 1,339 786 1,339 824 1,339 862 1,339 900 1,339 938 1,339 976 1,340 014 1,340 052
33 1,340 090 1,340 127 1,340 164 1,340 201 1,340 238 1,340 275 1,340 312 1,340 349 1,340 386 1,340 423
34 1,340 470 1,340 508 1,340 546 1,340 584 1,340 622 1,340 660 1,340 698 1,340 736 1,340 774 1,340 812
35 1,340 850 1,340 888 1,340 926 1,340 964 1,341 002 1,341 040 1,341 078 1,341 116 1,341 154 1,341 192
36 1,341 230 1,341 268 1,341 306 1,341 344 1,341 382 1,341 420 1,341 458 1,341 496 1,341 534 1,341 572
37 1,341 610 1,341 648 1,341 686 1,341 724 1,341 762 1,341 800 1,341 838 1,341 876 1,341 914 1,341 952
38 1,341 980 1,342 017 1,342 054 1,342 091 1,342 128 1,342 165 1,342 202 1,342 239 1,342 276 1,342 313
39 1,342 360 1,342 398 1,342 436 1,342 474 1,342 512 1,342 550 1,342 588 1,342 626 1,342 664 1,342 702
40 1,342 740 1,342 778 1,342 816 1,342 854 1,342 892 1,342 930 1,342 968 1,343 006 1,343 044 1,343 082
41 1,343 120 1,343 158 1,343 196 1,343 234 1,343 272 1,343 310 1,343 348 1,343 386 1,343 424 1,343 462
42 1,343 500 1,343 538 1,343 576 1,343 614 1,343 652 1,343 690 1,343 728 1,343 766 1,343 804 1,343 842
43 1,343 870 1,343 907 1,343 944 1,343 981 1,344 018 1,344 055 1,344 092 1,344 129 1,344 166 1,344 203
44 1,344 250 1,344 288 1,344 326 1,344 364 1,344 402 1,344 440 1,344 478 1,344 516 1,344 554 1,344 592
45 1,344 630 1,344 668 1,344 706 1,344 744 1,344 782 1,344 820 1,344 858 1,344 896 1,344 934 1,344 972
46 1,345 000 1,345 037 1,345 074 1,345 111 1,345 148 1,345 185 1,345 222 1,345 259 1,345 296 1,345 333
47 1,345 380 1,345 418 1,345 456 1,345 494 1,345 532 1,345 570 1,345 608 1,345 646 1,345 684 1,345 722
48 1,345 750 1,345 787 1,345 824 1,345 861 1,345 898 1,345 935 1,345 972 1,346 009 1,346 046 1,346 083
49 1,346 130 1,346 168 1,346 206 1,346 244 1,346 282 1,346 320 1,346 358 1,346 396 1,346 434 1,346 472
50 1,346 500 1,346 537 1,346 574 1,346 611 1,346 648 1,346 685 1,346 722 1,346 759 1,346 796 1,346 833

250
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253
Medical Biophysics with practical course - University textbook (scriptum)
Jakub Míšek, Marcel Veterník, Ján Jakuš, et al.

Published by Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava,

Martin, 2022. 253 numbered pages. 2nd revised, updated and extended (English) edition.
Printed in 2022 by DOLIS GOEN, Bratislava.

ISBN 978-80-8187-129-0
EAN 9788081871290