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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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Quantitative fluorescence microscopy techniques to study three-dimensional organisation of T-cell signalling molecules.
Chum, Tomáš ; Cebecauer, Marek (advisor) ; Lánský, Zdeněk (referee) ; Brameshuber, Mario (referee)
10 SUMMARY Proteins represent one of the basic building blocks of all organisms. To understand their function at the molecular level is one the critical goals of current biological, biochemical and biophysical research. It is important to characterise all aspects that affect the localisation of proteins into different compartments with specific functions, the dynamic structure of proteins and their role in multiprotein assemblies, because altering these properties can lead to various diseases. Most of the proteomic studies are nowadays performed using biochemical approaches that allow us to study multicellular organism or tissue at once. The disadvantage of these methods is complex preparation of sample and the need for a large number of cells, which leads to the loss of information at the molecular level and in individual cells. On the contrary, microscopy can provide rather detailed information about proteins of interest and at the level of a single cell. A variety of fluorescence microscopy methods in combination with recombinant DNA techniques were applied to elucidate subcellular localisation of transmembrane adaptor proteins (TRAPs) in human lymphocytes and their nanoscopic organisation at the plasma membrane. Linker of activation of T lymphocytes (LAT), phosphoprotein associated 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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