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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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Preparation of Specimen for Transmission Electron Microscopy using Freeze Substitution and Agitation
GRAHAMMER, Johannes
The quality of biological samples for Transmission Electron Microscopy prepared using High Pressure Freezing and Freeze Substitution with a novel agitation module invented by Helmuth Goldhammer and Siegfried Reipert was compared to Cells prepared without agitation and the Freeze Substitution protocol by Obornik et al. The amount of extraction was quantified via immunolabelling of RuBisCO inside the plastids of the Alveolate Chromera Velia Mechanical Damages were quantified by counting of obviously damaged and intact cells at low magnification
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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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