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Cryo-SEM and TEM of High Pressure Frozen Cells - Some Technical Contributions

Published online by Cambridge University Press:  02 July 2020

Paul Walther*
Affiliation:
Central Laboratory for Electron Microscopy, University of Ulm, D-89069, Ulm, Germanyhttp://www.uni-ulm.de/elektronenmikroskopie/http://www.nice.org.uk/page.aspx?o=43210
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Abstract

Imaging of fast frozen samples is the most direct approach for electron microscopy of biological specimen in a defined physiological state. It prevents chemical fixation and drying artifacts. High pressure freezing allows for ice-crystal-free cryo-fixation of tissue pieces up to a thickness of 200 urn and a diameter of 2 mm without prefixation. Such a frozen disc, however, is not directly amenable to electron microscopic observation: The structures of interest have to be made amenable to the electron beam, and the structures of interest must produce enough contrast to be recognized in the electron microscope. This can be achieved by freeze fracturing, cryo-sectioning or freeze substitution.

The figures show high pressure frozen bakers yeast saccharomyces cerevisiae in the cryo-SEM (Figures 1 and 2) and after freeze substitution in the TEM (Figure 3). For high pressure freezing either a Bal-Tec HPM 010 (Princ. of Liechtenstein; Figures 1 and 2), or a Wohlwend HPF (Wohlwend GmbH, Sennwald, Switzerland; Figure 3) were used.

Type
Cryoimmobilization, Freeze Substitution and Cryoem (Organized by S. Erlandsen)
Copyright
Copyright © Microscopy Society of America 2001

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References

Walther, P, Hentschel, J. (1989). Improved representation of cell surface structures by freeze substitution and backscattered electron imaging. Scanning Microsc 1989, 3, Supplement 3: 201211.Google ScholarPubMed

Walther, P, E, Wehrli, Hermann, R, M., Muller (1995) Double layer coating for high resolution low temperature SEM. J Microsc. 179,229237.CrossRefGoogle Scholar

Walther, P and Muller, M. (1999) Biological ultrastructure as revealed by high resolution cryo-SEM of blockfaces after cryo-sectioning. J. Microsc, 196 (3) 279287CrossRefGoogle Scholar

Zierold, K (2000) Heavy metal cytotoxicity studied by electron probe X-ray microanalysis of cultured rat hepatocytes. Toxicol in Vitro, 14, 557-63.CrossRefGoogle ScholarPubMed