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Focus techniques of optical measurement of 3D features
Macháček, Jan ; Honec, Peter (referee) ; Janáková, Ilona (advisor)
This thesis deals with optical distance measurement and 3D scene measurement using focusing techniques with focus on confocal microscopy, depth from focus and depth from defocus. Theoretical part of the thesis is about different approaches to depth map generation and also about micro image defocusing technique for measuring refractive index of transparent materials. Then the camera calibration for focused techniques is described. In the next part of the thesis is described experimentally verification of depth from focus and depth from defocus techniques. For the first technique are shown results of depth map generation and for the second technique is shown comparison between measured distance values and real distance values. Finally, the discussed techniques are compared and evaluated.
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Photosynthetic cell suspension cultures quantitative image data processing
Vlachynská, Alžběta ; Búzová,, Diana (referee) ; Červený,, Jan (advisor)
This work was carried out in collaboration with the Department of Adaptive Biotechnologies, Global Change Research Centre AS CR. It deals with the quantitative analysis of photosynthetic cell cultures. It uses images captured by a confocal fluorescent microscope to the automatic determining the number of cells in the sample. The work consists of a theoretical analysis, which briefly describes fluorescence and confocal microscopy. It also concisely introduces a microscope Leica TCS SP8 X, which I used to scan data. One capture is devoted to the theory of digital image processing. The second part deskribes the development of algorithm for processing 3D data and simplified algorithm for processing 2D data and its program implementations in a programming environment MATLAB R2013b. Grafical user interface is explained in detail. Done measurement are presented at the conclusion. It mentions compiled sample preparation protocol. The results of the program are compared with manual counting. Number of cells per 1 ml are determined by created program in samples of cell cultures Chenopodium rubrum (Cr) and Solanum lycopersicum (To).
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Software FLIM system with pulse white light laser in confocal microscopy
Grund, Pavel ; Odstrčilík, Jan (referee) ; Čmiel, Vratislav (advisor)
The theoretical part of this master's thesis is focused on research of confocal microscopy and FLIM method. There are a principles and types of confocal microscopy and the use of broad-spectrum laser as a basic light source of these microscopes. It gives what the FLIM method and its use not only in cell biology. The practical part thesis includes the acquisition of three sets of fluorescence intensity images with use of applications tunable pulsed laser, function TimeGate and detection of hybrid detectors. For practical elaboration of this thesis is in the software Fiji created a plugin, which is the source code in the Java programming language. The types of plugins and their uses are described in the third chapter of the thesis. This plugin including the graphical user interface in the form of the dialog box, proceses the fluorescence intensity images and creates a graphical representation of data showing the fluorescence lifetime, so called pseudocolor map.
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Study of cardiomyocytes viability using confocal microscopy
Chutný, Pavel ; Svoboda, Ondřej (referee) ; Čmiel, Vratislav (advisor)
This work describes the study of cardiomyocytes viability using confocal microscopy. The theoretic introduction covers the basic principle of fluorescence together with fluoresceins in cells biology. The struction of cardiomyocytes, their selection from the lively heart and the methods of viability examination is comprised in the next part. The third topic is about confocal mikroskopy and its posibilities in cardiomyocytes imaging. In next chapters are described the experiments and their evaluating.
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ANALYSES OF STRUCTURE OF COLLAGEN FIBRES IN ARTERIAL WALL USING MODERN METHODS OF LIGHT MICROSCOPY
Turčanová, Michaela ; Kochová, Petra (referee) ; Hampl, Aleš (referee) ; Burša, Jiří (advisor)
The doctoral thesis deals with the analysis of the arrangement of collagen fibres in arteries and their correct evaluation and use in structurally motivated constitutive models of the material. The first part of the work is focused on the literature search of mechanical properties of arteries and on an overview of available methods for the detection of waviness, orientation and dispersion of fibres. Most works identify fibre angles as additional parameter from mechanical tests and thus degrade the structural nature of the model. The second part describes an automatic algorithm that can evaluate the local directions of fibres and their scattering from images from a polarizing microscope and structurebased hyperelastic constitutive models. Furthermore, there is an emphasis on choosing the most appropriate imaging method based on fluorescence microscopy, which will help us to distinguish the waviness and scattering of fibres. In the next part of the thesis, two experiments on porcine arteries are presented in order to determine the influence of different magnitudes of biaxial deformation on fiber orientation and dispersion. The last part of the work presents the evaluated structural parameters for porcine and human aortas, which were analyzed not only under polarized light, but also under a laser scanning confocal microscope, thanks to which the waviness of the fibers and their global direction were obtained.
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Analysis of adherent cells confluency in 2D culture
Bracková, Michaela ; Čmiel, Vratislav (referee) ; Chmelíková, Larisa (advisor)
First part of this semester work dealing with the theoretical description of adherens cells, particularly structure and functions of this cells. Subsequently work is devoted to description of cell culturing, with mention of conditions which are necessary for cell culturing, following by substances which promote cell growth. Last part of theoretical research is concern with microscopy technique that is suitable for studying of adherent cells. Subsequently it is about fluorescence and confocal microscopes. Practical part dealing with cell culturing of adherent cells and the evaluation of realized experiments.
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Evaluation of mitochondrial activity in living cells using fluorescence methods
Fedorov, Vasilii ; Mézl, Martin (referee) ; Čmiel, Vratislav (advisor)
Monitoring mitochondrial dynamics can help in the therapeutic treatment of serious diseases and slowing down the aging process. This work was parted into a theoretical part and a practical part. In the theoretical part, research was carried out in the field of mitochondria and methods of activity evaluation. In the practical part in the Python environment, an automatic algorithm for evaluating mitochondrial activity based on the analysis of morphological signs was implemented. The algorithm included image preprocessing, localization of mitochondria, segmentation, and calculation of morphological features such as number of mitochondria, total area, average area, circularity index, eccentricity index. We were able to get and perform an analysis on 16 microscopic images and confirm the success of an algorithm that could be used for drug discovery and testing purposes.
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3D rekonštrukcia povrchu buniek hypokotylov Arabidopsis thaliana so zmenenými hladinami cytokinínov
Hubinský, Marcel
Plants are multicellular organisms, which tightly control their growth and morphogene-sis to successfully accomplish the final reproductive individual plant. At the cellular level, plant development consists of two main processes, cell proliferation and cell ex-pansion. Differences in the timing, rate and spacing of these developmental processes determine the final size and structure of plants organs, such as hypocotyl. Cytokinins (CKs) play a key role during many aspects of plant development, including hypocotyl development. We confirmed the important role of CK during expansion phase of hypocotyl cells. Hypocotyls with increased levels of CK growing under low light conditions were formed by elongated epidermal cells whereas hypocotyls lacking CK produced smaller cells. Using advanced confocal microscopy methods and transgenic plants with GFP-labeled cortical microtubules (proCaMV35S::MBD-GFP) enabled to design recon-struction of hypocotyl surface with altered levels of CK. The final 3D model of hypocotyl epidermal cells shed a light on detail morphological changes upon CK alterations
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Exprese proteinu NS5 viru klíšťové encefalitidy v lidských neurálních buňkách
JAKLOVÁ, Kateřina
This study focuses on the detection of tick-borne encephalitis virus (TBEV) NS5 protein in infected and NS5-transfected DAOY HTB-186 human neural cells. TBEV NS5 protein was shown to localize mainly on the membranes of the endoplasmic reticulum. An interesting finding was also nuclear localization, which is supported by the obtained data from both, confocal microscopy and subcellular fractionation.
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Fluorescent Methods in Research of Eukaryotic Cells
Chmelíková, Larisa ; Babula, Petr (referee) ; Pešl,, Martin (referee) ; Provazník, Ivo (advisor)
Tato práce zkoumá aplikaci fluorescenčních metod používaných v in vitro studiích v oblasti regenerace srdeční tkáně. Konfokální fluorescenční mikroskopie je vhodnou mikroskopickou technikou pro výzkum v této oblasti, protože umožňuje vizualizaci 3D struktur a distribuce buněk ve 3D modelech. Používané fluorescenční markery by měly být dlouhodobě stabilní, biokompatibilní a netoxické pro živé buňky. V současné době je použití nanočástic jako superparamagnetické nanočástice oxidu železa (SPION) velmi populární; velké množství studií ukazuje, že jsou vhodné pro dlouhodobé experimenty. Tento výzkum využívá superparamagnetické maghemitové nanočástice svázaným rhodaminem na jejich povrchu (SAMN-R) a popisuje jejích excitační a emisní spektrum, velikost a lokalizaci vbuňkách. Stanovení toxicity bylo provedeno měřením reaktivních forem kyslíku (ROS) a nekvantitativním měřením pomocí fluorescenční mikroskopie bylo zjištěno, že hodnota dávky 20 µg·cm-2 je optimální pro aplikaci na živé buňky. Dále byl zkoumán vliv aplikace SAMN-R na buněčnou proliferaci a motilitu, kdy ve studii buněčné proliferace a scratch assay byla použita buněčná linie fibroblastů 3T3. Poté byla studována migrace jednotlivých buněk s použitím mezenchymálních kmenových buněk (MSCs), izolovaných zlidské tukové tkáně. Následná statistická analýza nepotvrdila, že by aplikace SAMN-R měla významný vliv na buněčnou proliferaci, kolektivní migraci nebo na migraci jednotlivých buněk. Lze tedy předpokládat, že SAMN-R jsou vhodným fluorescenčním markerem pro výzkum živých buněk, včetně experimentů voblasti regenerace tkáně. MSC buňky izolované z tukové tkáně mají velký potenciál v regeneraci srdeční tkáně. Jejich interakce s buněčnou linií srdečních svalových buněk HL-1 byly studovány pomocí scratch assay, kdy se tento model jeví jako nadějný a vhodný pro studium buněčných kontaktů a jejich roli přiregeneraci buněk.
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