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Membrane potential measurement with voltage sensitive dyes in fluorescence microscopy
Tkáč, Jan ; Janoušek, Oto (referee) ; Čmiel, Vratislav (advisor)
The aim of this work is to make a literature search in the measurement of membrane voltage using voltage-sensitive dyes and suggest a method for measuring the membrane voltage on the available cells using the voltage-sensitive dye di 4 ANEPPS and its further implementation. The work contains an introduction to electrophysiology of cells, and explains typical fluorescence characteristics. The thesis contains the description of a fluorescence microscope. The document presents characteristics of voltage-sensitive dyes and their distribution. A large part of the work describes the implementation and measurement of the experiment. The document also includes different methods for measuring and processing of all results.
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Membrane potential measurement with voltage sensitive dyes in confocal microscopy
Heczková, Monika ; Sekora, Jiří (referee) ; Čmiel, Vratislav (advisor)
The aim of this work is to make a literature search in the measurement of membrane voltage using the voltage-sensitive dyes and suggest a method of measuring the membrane voltage on the available cells using the voltage-sensitive dye Di-4-ANEPPS and RH237. The work contains an introduction to electrophysiology of cell, explains fluorescence and typical fluorescence characteristics. The thesis contains the description of a fluorescence microscope. The document was largely devoted to characterization and distribution of voltage-sensitive dyes. The output of a design solution is a real experiment.
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Simulation of action potentials and membrane currents by solving Hodgkin and Huxley equations
Bugnerová, Pavla ; Provazník, Ivo (referee) ; Chmelíková, Larisa (advisor)
The thesis deals with the analysis of the resting membrane potential and action potential mainly on the cell membrane of cardiac cells. Attention is given to the electrical schematic layout of the cell membrane and distribution of current part on the main components. The basis for the creation of a mathematical model and simulation is the differential equations of Hodgkin and Huxley. To the description them of calcium and potassium channel leakage from other models are added. The project explores the possibilities of reproducing the known development of membrane currents in two basic modes (voltage clamp and current clamp). All in user interface MATLAB GUI.
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Monitoring of mitochondria in living cells using fluorescence methods
Taliánová, Eliška ; Čejková, Darina (referee) ; Čmiel, Vratislav (advisor)
This bachelor thesis deals with the topic of monitoring mitochondria and evaluating their activity. The structure and function of cellular mitochondria are briefly discussed. Contains a list of commonly available fluorescent dyes. It investigates the possibilities of influencing mitochondria in vitro. It deals with the influence of mitochondrial membrane potential by means of various concentrations of glucose and subsequently evaluates mitochondrial activity using the dye JC-10.
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Monitoring of mitochondria in living cells using fluorescence methods
Taliánová, Eliška ; Čejková, Darina (referee) ; Čmiel, Vratislav (advisor)
This bachelor thesis deals with the topic of monitoring mitochondria and evaluating their activity. The structure and function of cellular mitochondria are briefly discussed. Contains a list of commonly available fluorescent dyes. It investigates the possibilities of influencing mitochondria in vitro. It deals with the influence of mitochondrial membrane potential by means of various concentrations of glucose and subsequently evaluates mitochondrial activity using the dye JC-10.
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Membrane potential measurement with voltage sensitive dyes
Votavová, Barbora ; Janoušek, Oto (referee) ; Čmiel, Vratislav (advisor)
The aim of this work is to realize measurements of membrane potential with voltagesensitive dye Di-4-ANEPPS and the data processed and analyzed. The work includes theoretical basis in the form of electrophysiology animal cells, explains fluorescence and describes the fluorescence microscope. The document is largely devoted to the characterization and distribution of voltage-sensitive dyes (VSD). The practical part deals with the various components necessary to perform the experiment as a pulse generator, high-speed camera and camera’s acquisition and describes experiment. The conclusion will be compared with results from theoretical assumptions.
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The comparison of the performace of selected carbocyanine dyes in fluorescent probing of yeast cell membrane potential.
Mudroňová, Kateřina ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
The membrane potential is one of the most important parameters of the living cell. It can be measured using carbocyanine fluorescent probes. In this thesis we examined parameters of several dyes of this family. For further experiments three of them were chosen - diOC3(3), diIC1(3) a diIC2(5) as a supplement to diSC3(3) and diSC3(5), which represent standard probes used at biophysical department of Institut of Physics. We compared the rates of their accumulation in S. cerevisiae cells to determine if they were MDR pumps' substrates. The other goal of this work was to decide whether the results obtained using different probes are equivalent and to determine if the presence of a probe affects the spectral characteristics of another. For this purpose we have chosen diSC3(3) and diSC3(5). With those dyes we examined the influence of the acidification on membrane potencial of the yeast S. cerevisiae. We showed that the information on depolarization obtained using both probes were matching very well.
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The comparison of the performace of carbocyanine dyes disC3(3) a diSC3(5) in fluorescent probing of yeast cell membrane potential.
Matunová, Petra ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
Membrane potential represents a voltage across a membrane and it is an important parameter that helps to describe processes in cells. Carbocyanine fluorescent probes diS-C3(3) and diS-C3(5), for which a common short chemical name 3,3'- dipropylthiadicarbocyanine iodide is used, are suitable for monitoring membrane potential changes of cells in which microelectrodes can not be used because of a small size of the cells. These changes can be measured on the scale of mV. A spectral analysis of cell suspensions containing a fluorescent probe makes it possible to determine the ratio of extracellular and intracellular concentrations of the probe. Using it we can calculate the value of membrane potential changes which can be induced by an outer stimulus. This Bc. thesis presents a comparison of the rate of accumulation of the above mentioned fluorescent probes in yeast cells, as well as experiments aimed for studying an inuence of different substances and their various concentrations on free and bound component of the dye.
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Response of plasma membrane potential and intracellular pH of yeast to changes in the concentration of extracellular K+
Babuka, David ; Plášek, Jaromír (advisor) ; Heřman, Petr (referee)
The bachelor thesis is focused on studying yeast cells and their response to various external conditions. Main focus was on the study of intracellular pH and membrane potential change under the condition of varying extracellular concentration of K+ ions. In particular we studied to what extent are the yeast cells able to compensate these changes. The ability of yeasts to resist the changes of external pH of the cell medium was studied in an experiment complementary to the measurements of intracellular pH. To measure the intracellular pH a genetically encoded fluorescent probe ratiometric pHluorin was used and to measure the changes of membrane potential a fluorescent probe diS-C3(3) was used. Also we successfully applied a method of synchronously scanned fluorescence to supress the cell autofluorescence.
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The effect of antimicrobial peptides on Saccharomyces cerevisiae and other yeast species
Makarova, Anna-Marie ; Hodek, Petr (advisor) ; Malcová, Ivana (referee)
The increased use of antibiotics, antifungal agents and disinfectants in the last decades has resulted in development of microbial resistance to these drugs. Candida species are the fourth most common cause of hospital-acquired bloodstream infection and kill 40% of those patients. Natural antimicrobial peptides are promising candidates for the development of new agents to treat yeast and bacterial infections, as their presumed mechanism of action differs significantly from the mechanism of action of current drugs. This work is focused on several peptides isolated from the venom of wild bees and their synthetic analogues and the identification of the most effective ones against non-pathogenic Saccharomyces cerevisiae and several pathogenic Candida species. Antifungal activity of eight cationic antimicrobial peptides was tested and compared under various conditions. The overall susceptibility of pathogenic yeast species to currently used antifungal drugs and the antimicrobial peptides was screened with the aim to identify potential synergistic and species-specific effects. The effect of antimicrobial peptides on membrane potential was measured by a fluorescent probe (diS-C3(3)), and the relative hyperpolarization of plasma membrane was shown for each peptide. The effect of antimicrobial peptides on...
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