The implementati edge of the composition and electrical properties of biological tissues also requires in-depth knowledge in the field of bioimpedance instrumentation and methods design. Biological tissues have a very complex composition whose structure is, as a rule, not homogeneous in isotropic, but is an array of a large number of structures with different electrical properties that perform various functions in closed biological systems. The electrical impedance of biological tissues (bioimpedance) as a material property depends on many physiological, structural and geometric properties of tissues and is used in many fields as an difference indicator. In the introductory part of the thesis, I devoted a few words to the description of the basic properties of cells in biological tissues, both physiological and electrical. I also listed and described the basic parameters that we usually use to describe and evaluate electrical properties of biological tissues. More information we have about the properties of the DUT (device under test or biological tissue) and the influential parameters, more input data for the measurement and the understanding of the results we have. In the research and medical field of bioimpedance measurements, simple, precise and above all reliable measurements are required, which are enabled by expensive and powerful impedance analyzers and LCR meters with aditional front-end. Their weakness is that they are usually too universal, stationary and unflexible, and for some medical applications and research often useless. In the thesis, I analyze, research and develop a methodology for measuring bioimpedance with portable, adaptable and medically safe systems, which are useful primarily for research purposes. To summarize, the goal of the thesis is the development of portable systems and methods for measuring bioimpedance. The thesis consists of three separate thematic sections that capture the measurement instrumentation, the measurement methods and their use in the biomedical research field. Each section consists of a theoretical introduction, a description of the problem, objectives and the presentation of the obtained results. In the first part of the thesis, I analyzed instrumentation, signal capturing and data processing. I started the theoretical part with a general description of the concept of electrical impedance, and continued with the description and analysis of the most commonly used measurement techniques. Special attention was paid to single sine technique and the reconstruction of the measurement results with lock-in amplifiers. They are based on phase sensitive detection, which has two basic operations: synchronous demodulation and filtering. I also presented the advantages and disadvantages of the multi sine technique, which is mainly used in measuring the impedance of dynamic materials with rapidly changing properties. At the end of the theoretical introduction, I also presented the basic components of the instrumentation for determining bioimpedance. The use of stationary impedance analyzers with large dimensions and high price is not always acceptable and affordable, therefore, portable and adjustable measuring systems are more acceptable for universal use. In the development of portable systems, it is reasonable to separate the measuring part from the processing one, in that way, the device is constructed for lower energy consumption, faster measurement performance, easier storage and more detailed later processing of the measured signals. In the continuation of the first section of thesis under the results column, I first introduced the guidelines for the development of portable, cheap and battery-powered bioimpedance measurement systems based on small microcontrollers. I have developed a simple measurement system that has a separate measuring part from the processing part, which allows for lower energy consumption, easier storage of data and more detailed processing of the measured signals. I used the WiFi communication protocol to transfer
data to portable devices. I analyzed the importance of individual components and evaluated the
uncertainties of the developed system through testing. It turns out that this kind of performance
contributes to the simplicity and cheapness of the device itself, but it loses in terms of precision and reliability of measurements. The key reasons are the capacity of the microcontroller and the absence of an excitation source. Based on the analysis, I decided to develop a voltage-controlled constant current source. The reason for this is the precise setting and assurance that the measurement object is excited by a safe and constant current over the entire frequency range of measurements, regardless of the value of the impedance of the DUT. I focused on the solution based on Howland's circuit with single power supply. For the evaluation of the quality and accuracy of the source, I carried out an analysis, whichcovered the influence of parasitic capacitance on the output parameters. It is known that the parasitic capacitance can not be completely avoided, but with an optimaly designed circuit board, their influence can be reduced. Besides the measurement part, I also investigated the processing part, especially the
reconstruction of the measurement results. It turned out that an appropriate method for reconstructionof the measured signals within the processing part is a phase-sensitive detection and associated frequency response analyzer, which effectively separates the signals of small amplitudes from noise. For the correct reconstruction of the amplitude and phase, they need a reference signal. I paid special mattention to evaluating the effect of frequency synchronization and averaging within the frequency response analyzers. I analyzed the influence of various parameters in the reconstruction and calculation of bioimpedance. I started with the non-synchronization of the reference and measured signals, and continued with the analysis and estimation of the consequences of the deviations of the frequency of the reference signal and the connection with the phase angle between the measured signals, the number of captured periods and different values of the signal-noise parameter. In the end, I checked all the theoretical assumptions with the help of experimental analysis. It turns out that in the case of an ideal
sinusoidal excitation signal and a zero phase angle between the measured current and voltage signals (ideal resistivity character of DUT), the non-synchronization to the calculation of the impedance is not affected. With the increase of the phase angle and the deviation of the frequency, the error is linearly increasing. In the last part of the first section, on the basis of the performed research and developeded components, I designed the concept of a bioimpedance measurement system with platform RedPitaya which is based on FPGA technology. The core of the measuring system is the RedPitaya measuring platform with added custom hardware or an interface for measuring bioimpedance parameters. System also has a separate measuring part from the processor part and its specialty is that it is adaptable to different applications in terms of the frequency range of measurements, the dynamic range of measurements, the possibility of stimulating the measured object with any form of excitation signal, multiple electrode measurement, resolution, speed, etc. I also implemented frequency response analyzer with software in the processing part. In addition to the implementation I introduced the three-reference calibration method and ultimately evaluated the measurement uncertainties for the magnitude and phase of bioimpedance. In the second part of the thesis, I dealt more closely with measurement methods and upgrading these with the usage of a 'floating conductive electrode'. I started the theoretical introduction by describing the difference between the concepts of mutual and trans-impedance, and continued with the sensitivity of electrode systems to small changes in complex conductivity of bioimpedance measurements. In general, the greater the absolute value of the sensitivity of a small volumetric contribution, the greater the contribution to the total measured impedance. The negative value of the sensitivity of a small volum contribution, however, means that the increase in resistivity only results in a decrease in the total
measured impedance. The placement and size of electrodes, both exciting and measuring, is very
important in measuring bioimpedance. This directly influences the sensitivity and depth of
measurements. By increasing the distance between the electrodes we increase the depth of measurement while changing the dimensions of the electrodes changes the volume sensitivity. I also analyzed measurement methods, where I summarized the characteristics of measuring bioimpedance with the two, three and four electrode systems. At the end of the theoretical part, I also presented the solution for geometric models calculating using the numerical methods.
The sensitivity analysis showed that the area close to and around the excitation electrodes is more sensitive to the overall measurement than the areas far away. In certain cases, the electrical properties of the measured object in the limited field of measurement between the electrodes are interesting, and also the contribution of this to the result of the total measurement. Moreover, in some cases, it is required to detect changes in electrical properties in the local measuring range. It is usually sufficient to move the electrodes to the desired area, but then only the current impedance value is measured, and the value before the occurrence of the change is not known. As an appropriate and easy solution to these problems I presented the upgrade of a 4-electrode measurement with an additional inactive conductive floating
electrode. The goal of this is to increase the local perception of changes in electrical properties. An additional electrode has the task of redistributing the current. In the results chapter, I presented the basic principle of upgrading existing methods with floating conductive electrodes. Using numerical simulations, I showed the action and confirmed the assumption that with the help of an added conductive electrode, it is possible to deduce the depth and electrical properties of local changes. I continued with the simulation of the use of floating electrode as a scan electrode for detecting local changes. Finally, using the 3D model, I have demonstrated the possible use in the field of research. In the third part of the thesis, I adjusted developed measurement instrumentation by introducing the interface and used it in a research application in the field of excitation of nerve tissues. In the theoretical introduction, I roughly presented the field of excitation of nerve tissue. I've raised features that must be considered when choosing an excitation pulse. These are: the relationship between the amplitude and duration, between the amplitude and the distance of the electrodes from the nerve tissue and between the amplitude and the width of the pulse. Furthermore, I analyzed the different types of electrodes used
in the excitation field. Particular attention should be paid to the biocompatibility of electrodes with tissue. I also analyzed in detail the problem of polarization due to the contact of the electrode and biological tissue. Because of the different nature of the currents at the contact between the electrode and the biological tissue, there are reactions that allow the conversion between electrons and ions. Immediately after contact with the tissue and moisture, a so-called double layer is created on the surface of the electrode, and within this the electron transfer occurs. In addition to the capacitance-specific phenomena at the electrode-electrolyte contact, there are also electrochemical or faradayic processes, where oxidation and reduction reactions take place. I have also described typical ways of characterizing electrodes and equivalent circuits by which we can model and optimize electrode-tissue contact based on the results of measurements. Existing systems for such characterization are usually separate units that need to be adapted to each other and perform individual measurements separately. This is especially evident when the stimulation and response measurement processes are separated, since in the meantime the situation can be changed and can no longer be recorded later. As a solution, I introduced a adjustable system with a built-in interface that allows simultaneous optional excitation and measurement of the voltage response and the calculation of bioimpedance. The exciting part is more important because the mechanism and shape and the power of the excitation signal influence the efficiency, selectivity and safety of stimulation. Inside
the chamber used to excite the isolated nervous tissue, there are platinum electrodes, which, when contacted with biological tissue, result in the formation of a double layer and other electrochemical reactions due to the conversion between electrons and ions. It turns out that a two-electrode measurement is more suitable for monitoring electrodes. Based on the research I developed a stimulation and measurement system and prepared the procedure of selective stimulation of the nervous tissue with a quasi-trapezoidal pulse. I compared the difference in the response between the bipolar and the polar excitation pulse. I recorded both measurement of the response and bioimpedance with a two-electrode and a four-electrode placement, and the results were compared with each other. With proper excitation, the reactions can be partially restricted to ensure their reversibility, thereby preventing possible damage to the excited tissue and electrodes. In addition to the response measurements, I sensed the compound action potential with the system and finally composed the bioimpedance spectrum in the wider frequency
range. I originally showed the results in Bode diagram, and then in Wessels diagram, where based on the characteristic course of the graph I concluded about the contents of the elements in the equivalent circuit. The data obtained in experiment are a good basis for further study and optimization of stimulation mechanisms.