High pressure freezing and freeze substitution of Schizosaccharomyces pombe and Saccharomyces cerevisiae for TEM.
Authors
Murray, Stephen MAffiliation
TEM Service Facility, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom.Issue Date
2008
Metadata
Show full item recordAbstract
The use of standard room temperature chemical fixation protocols for the ultrastructural preservation of yeast and subsequent observation under the electron microscope is fraught with difficulties. Many protocols require the use of enzymatic digestion of the cell wall in order to facilitate the entry of fixatives into the cell interior. Others rely on the use of permanganate-based fixative solutions, which whilst enabling overall preservation of the cell, does require multiple centrifugation, washing, and resuspension steps. This often results in the significant loss of sample volume whilst the use of permanganate can cause extraction of cytoplasmic components. The use of low temperature techniques and in particular high pressure freezing (HPF) and freeze substitution (FS) overcomes many of these problems. With the recent advances in cryotechnologies and in particular the development of commercially available equipment such as the high pressure freezer, the level of ultrastructural preservation attainable in electron microscopy has increased markedly. It is now possible to capture dynamic time sensitive events and to place them in their ultrastructural context with a level of resolution which at the present time can only be achieved with electron microscopy.Citation
High pressure freezing and freeze substitution of Schizosaccharomyces pombe and Saccharomyces cerevisiae for TEM. 2008, 88:3-17 Methods Cell Biol.Journal
Methods in Cell BiologyDOI
10.1016/S0091-679X(08)00401-9PubMed ID
18617025Type
Book chapterLanguage
enISSN
0091-679Xae974a485f413a2113503eed53cd6c53
10.1016/S0091-679X(08)00401-9