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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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Cell Viability Measurement
Pelc, Pavel ; Janoušek, Oto (referee) ; Čmiel, Vratislav (advisor)
This work discusses the viability of cells. It describes the principle of fluorescence, fluorescent methods, fluorescent microskopy and the method of dyeing of the cells with a special kit that contains calcein AM and ethidium homodimer dyes. Further it describes the program for analysis and evaluation of viability on two different cellular cultures.
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Anisotropy techniques in study of cytoplasm
Sýkorová, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The main goal of this thesis was to compare experiments using time-resolved anisotropy and steady-state anisotropy for measuring in bacteria strain Cupriavidus necator. Fluorescent probe for anisotropy imaging was chosen BCECF_AM, which is derivate of fluorescein. Using experiment in system glycerol/water with fluorescein, anisotropy has been verified and calculated molecular hydrodynamic volume of a single fluorescein molecule, which approximately corresponded with real value. By using fluorescence imaging anisotropy microscopy, images and values of average anisotropy in cells were taken. Images of living cells (bacteria) of CN H16 and mutant CN PHB-4 showed differences, mainly in the uniformity of the inside environment.
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Natural drugs in cancer treatment and their cytotoxicity
Hájková, Tereza ; Raudenská, Martina (referee) ; Provazník, Ivo (advisor)
The thesis deals with the natural substances in context with the cancer disease. The natural substances have a positive effect on the human organism and they are able to influence the viability and the growth of the cancer cells. The main mechanical device is to influence the mechanisms needed to start the apoptosis of the cancer cells and stopping further proliferation. The cancer cell lines utilization in the cancer disease is discussed in the thesis too. The thesis states common methods of determining the natural substances cytotoxicity. For the experimental part of the thesis it was chosen the MTT test method and the xCELLigence system for monitoring in real time. The mechanical device of the tested substance capsaicin in application on the prostate cell lines, tumorous PC3 and nontumorous PNT1A influence will be observed within the experimental part of the thesis.
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Cell layer cultivation in the microfluidic system
Kachan, Ksenia ; Svoboda, Ondřej (referee) ; Chmelíková, Larisa (advisor)
The theoretical part of this thesis describes the principles of cell culture in vitro and modern in vitro models for endothelial cell layer cultivation. It also describes the principles of confocal and fluorescence microscopy. The practical part is dedicated to working with cell cultures and realization of experiment with cell cultivation in microfluidic system. Cell layer was photographed using laser confocal scanning microscope Leica TCS SP8 X during the entire experiment. For processing and analysis of the obtained images, an algorithm in the software MATLAB was proposed.
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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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Fluorescence imaging techniques in multimodal holographic microscope
Vašíček, David ; Procházková, Jana (referee) ; Čolláková, Jana (advisor)
The diploma thesis deals with the registration of images taken with the multimodal holographic microscope (MHM). The summary covers the fluorescent and holographic microscopy, and the multimodal holographic microscope combining both these microscopy types. Every pair of the images needs to be aligned in order to gain new information by combining both image types. The thesis contains an algorithm that registers images by phase correlation as well as a process created in MATLAB in accordance with the algorithm. The most important procedure parameters’ influence on the registration success is described and the results are annotated.
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Determination of the area ratio of tubular and surface membranes in cardiomyocytes
Hluší, Veronika ; Čmiel, Vratislav (referee) ; Chmelíková, Larisa (advisor)
This work contains a literature search regarding the function and structure of the tubular system of cardiomyocytes. Another theory includes the basic principle of fluorescence and a description of the fluorescence and confocal microscope. Next chapter describe the principle of isolation and subsequent staining of cardiomyocytes to obtain images of the tubular system is described. Next step is the implementation of a method for segmentation of the membrane and tubular system of cardiac cells and the application of this method to the obtained data.
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Study of fluorescence lifetime and spectral changes of nanoparticles in cell biology
Pelc, Pavel ; Janoušek, Oto (referee) ; Čmiel, Vratislav (advisor)
This work deals with the study of fluorescence lifetime and spectral changes of nanoparticles in cell biology. It describes the principle of fluorescence, fluorescence microscopy and laser confocal microscope Leica TCS SP8. The classic FLIM method, the Lambda Square mapping and the division of nanoparticles are introduced there. In the practical part, the created program for the evaluation of fluorescence lifetime and spectral changes is described. The program can show two-dimensional lambda maps, the fluorescence lifetime and spectral shift in the space area. In the final part of the thesis, an experiment with rhodamine nanoparticles is carried out and it is evaluated using the created program and then discussed.
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Analysis of neurite directionality
Plišková, Diana ; Čmiel, Vratislav (referee) ; Odstrčilík, Jan (advisor)
Práca je zameraná na navrhnutie vhodnej metódy analýzy smerovosti neuritov. Využité boli snímky neurónov z fluorescenčnej mikroskopie. Pred samotnou segmentáciou bolo potrebné snímky predspracovať, pričom sa postupne využila úprava kontrastu, ostrenie a adaptívna filtrácia pomocou Weinerovského filtru. Jednotlivé návrhy metód segmentácie pozostávali z prostého prahovania, narastaním oblastí a využitím morfologických operácií. Následná analýza smerovosti využívala smer gradientov v obraze. Navrhnutá metóda bola využitá aj ako klasifikátor, ktorý dokázal rozdeliť jednotlivé snímky do skupín podľa smeru rastu.
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