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Electron cryo-microscopy techniques in biological research and nanotechnologies
Mistríková, Veronika ; Bednár, Jan (advisor) ; Nebesářová, Jana (referee) ; Benada, Oldřich (referee)
Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...
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Electron cryo-microscopy techniques in biological research and nanotechnologies
Mistríková, Veronika ; Bednár, Jan (advisor) ; Nebesářová, Jana (referee) ; Benada, Oldřich (referee)
Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...
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Comparison of freeze fracture images of mixed bacterial/yeast biofilm in cryo-SEM with high pressure freezing fixation
Hrubanová, Kamila ; Nebesářová, Jana ; Růžička, F. ; Krzyžánek, Vladislav
Microscopic organisms include bacteria and yeasts have been studied in this project. Besides the planktonic way of living, microbes are able to adhere to surfaces or interfaces and to form organized communities, a so-called biofilm, which are embedded in a matrix of extracellular polymeric substances that they produce; visualization and quantification of this microscopic formation is the main goal of this study. In medicine the biofilm formation allows microorganisms to colonize the surface of implants and it also protects the microbial cells from attacks by the immunity system as well as from the effect of antibiotics. Therefore, the biofilm is considered to be important virulence factor in these microorganisms. The characteristic features of the biofilm infections, especially high resistance to antifungal agents, complicate therapy. Understanding of the biofilm structure can contribute to understanding the biofilmformation and basic biochemical mechanisms underlying this process. It may help to develop more efficient treatment strategy for biofilm infection.
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