Plunge Freezing for Electron Cryomicroscopy
Abstract
Aqueous biological samples must be "preserved" (stabilized) before they can be placed in the high vacuum of an electron microscope. Among the various approaches that have been developed, plunge freezing maintains the sample in the most native state and is therefore the method of choice when possible. Plunge freezing for standard electron cryomicroscopy applications proceeds by spreading the sample into a thin film across an EM grid and then rapidly submerging it in a cryogen (usually liquid ethane), but success depends critically on the properties of the grid and sample, the production of a uniformly thin film, the temperature and nature of the cryogen, and the plunging conditions. This chapter reviews plunge-freezing principles, techniques, instrumentation, common problems, and safety considerations.
Additional Information
© 2010 Elsevier Inc. Available online 29 September 2010. We wish to thank our colleagues, Steve Coyle and John Hunt of Gatan, Inc., Mark Ladinsky, Elitza Tocheva, Ariane Briegel, and Martin Pilhofer at Caltech, who proofread the manuscript. FEI, Inc.; Gatan, Inc.; Leica Microsystems, Inc.; and Electron Microscopy Sciences supplied images and data. Guenter Resch, IMP/IMBA Electron Microscopy Facility, Stefan Westermann and Angela Pickl-Herk, at the Max F. Perutz Laboratories in Vienna, Austria, provided specimens, images, and protocols from their experience using the Leica EM GP. Chen Xu, Rosentiel Basic Medical Sciences Research Center, Waltham, MA provided the image for Fig. 3.2B. This work was supported in part by NIH grant P01 GM066521 to G. J. J., the Beckman Institute at Caltech, and gifts to Caltech from the Gordon and Betty Moore Foundation and Agouron Institute. Finally, a special thank you to Ted and Chris Pella, Pella, Inc., for their bequest to the Jensen laboratory tomography database project.Additional details
- Eprint ID
- 22556
- Resolver ID
- CaltechAUTHORS:20110228-143852960
- P01 GM066521
- NIH
- Caltech Beckman Institute
- Gordon and Betty Moore Foundation
- Agouron Institute
- Created
-
2011-03-03Created from EPrint's datestamp field
- Updated
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2021-11-09Created from EPrint's last_modified field
- Series Name
- Methods in Enzymology
- Series Volume or Issue Number
- 481