Abstract
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 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 breaking the sub-nanometer limit.
These methods are presented in this thesis. The budding yeast Saccharomyces cerevisiae processed by high-presure freezing a freeze substitution was selected as a biological object for both fine ultrastructural and immunocytochemical studies using conventional transmission electron microscopy; the liposomes and other types of vesicular structures vitrified by plunge freezing were selected as nanoobjects for electron cryo-microscopic studies.