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J Struct Biol. 2013 Jan;181(1):77-81. doi: 10.1016/j.jsb.2012.10.005. Epub 2012 Oct 16.

Vitrification of thick samples for soft X-ray cryo-tomography by high pressure freezing.

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  • 1Dept. of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel.

Abstract

Soft X-ray cryo-microscopy (cryo-XT) offers an ideal complement to electron cryo-microscopy (cryo-EM). Cryo-XT is applicable to samples more than an order of magnitude thicker than cryo-EM, albeit at a more modest resolution of tens of nanometers. Furthermore, the natural contrast obtained in the "water-window" by differential absorption by organic matter vs water yields detailed images of organelles, membranes, protein complexes, and other cellular components. Cryo-XT is thus ideally suited for tomography of eukaryotic cells. The increase in sample thickness places more stringent demands on sample preparation, however. The standard method for cryo-EM, i.e., plunging to a cryogenic fluid such as liquid ethane, is no longer ideally suited to obtain vitrification of thick samples for cryo-XT. High pressure freezing is an alternative approach, most closely associated with freeze-substitution and embedding, or with electron cryo-microscopy of vitreous sections (CEMOVIS). We show here that high pressure freezing can be adapted to soft X-ray tomography of whole vitrified samples, yielding a highly reliable method that avoids crystallization artifacts and potentially offers improved imaging conditions in samples not amenable to plunge-freezing.

Copyright © 2012 Elsevier Inc. All rights reserved.

[PubMed - indexed for MEDLINE]
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