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Optical microscopy to study the role of cytoskeleton in cell locomotion and virus trafficking
Difato, Francesco ; Forstová, Jitka (advisor) ; Hozák, Pavel (referee) ; Plášek, Jaromír (referee)
3. General conclusions 150 The interest in optical microscopy is constanly growing, mainly because of its unique features in examining biological systems in four dimensions (x-y-z-t)1 . The work presented here was focused on biological applications of optical microscopy by exploring and improving the spatial and temporal resolution performances and by futher developing optical tools for manipulating biological samples. First, I studied the resolution performances of the system in the three dimensional space and I contributed in improving the experimental spatial resolution of microscope by applying deconvolution. In this respect, theoretical modelling can characterize the image formation process of the microscope, but only experimental measurement of the PSF can quantify the limitations of the real system. Indeed, experimental PSF presents shape assymetry due to spherical aberrations introduced by optical elements, while theoretical PSF is symmetric and account only for the resolution limits of an ideal imaging system. The disadvantage of experimental PSF is that could be corrupted by noise, otherwise deconvolution with the theoretical PSF offer only a qualitative improvement of the image, because the introduced artefacts cannot be quantified. Deconvolution of the acquired data with experimental PSF...
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Comprehensive characterization of cellular suspensions by means of elastic light scattering
Macháň, Radek ; Štěpánek, Josef (advisor) ; Plášek, Jaromír (referee)
Studies of yeast Saccharomyces cerevisiae - a model eukaryot organism - often require determining the number of cells in a suspension. One of the methods often used for studying cell suspensions is the measuring of elastic scattering of light passing through the suspensions. Usual output of those measurements is the angular dependency of the intensity of scattered light, which bears information about the size of scatterers. This thesis studies time-dependencies of the intensity of scattered light and relationship of their statistical properties to the number of scatterers in a unit volume. For this purpose a simple model was proposed, which describes the connection between this quantity for a monodisperse suspension and the dependency of standard deviation of time-behaviour of the scattered intensity on the mean value of the same time-behaviour. An apparatus for computer controlled measurements of angular and timedependencies of scattered light intensity was completed for carrying out the experiments. Elastic scattering was measured on suspensions of yeast Saccharomyces cerevisiae and a model system (suspensions of 8m polymer particles). Experimental data were interpreted within the proposed model. Although yeast suspensions cannot be regarded as monodisperse, experimental data were in a good agreement with...
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Computational methods in single molecule localization microscopy
Ovesný, Martin ; Hagen, Guy Michael (advisor) ; Plášek, Jaromír (referee) ; Fliegel, Karel (referee)
Computational methods in single molecule localization microscopy Abstract Fluorescence microscopy is one of the chief tools used in biomedical research as it is a non invasive, non destructive, and highly specific imaging method. Unfortunately, an optical microscope is a diffraction limited system. Maximum achievable spatial resolution is approximately 250 nm laterally and 500 nm axially. Since most of the structures in cells researchers are interested in are smaller than that, increasing resolution is of prime importance. In recent years, several methods for imaging beyond the diffraction barrier have been developed. One of them is single molecule localization microscopy, a powerful method reported to resolve details as small as 5 nm. This approach to fluorescence microscopy is very computationally intensive. Developing methods to analyze single molecule data and to obtain super-resolution images are the topics of this thesis. In localization microscopy, a super-resolution image is reconstructed from a long sequence of conventional images of sparsely distributed single photoswitchable molecules that need to be sys- tematically localized with sub-diffraction precision. We designed, implemented, and experimentally verified a set of methods for automated processing, analysis and visualization of data acquired...
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