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Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material

  • Special Issue: New/Emerging Techniques in Biological Microscopy
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Abstract

This article presents the best current practices for preparation of biological samples for examination as thin sections in an electron microscope. The historical development of fixation, dehydration, and embedding procedures for biological materials are reviewed for both conventional and low temperature methods. Conventional procedures for processing cells and tissues are usually done over days and often produce distortions, extractions, and other artifacts that are not acceptable for today’s structural biology standards. High-pressure freezing and freeze substitution can minimize some of these artifacts. New methods that reduce the times for freeze substitution and resin embedding to a few hours are discussed as well as a new rapid room temperature method for preparing cells for on-section immunolabeling without the use of aldehyde fixatives.

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Abbreviations

CF:

Conventional fixation

CLEM:

Correlative light and electron microscopy

EM:

Electron microscopy

FS:

Freeze substitution

HPF:

High-pressure freezing

LN2 :

Liquid nitrogen

LRW:

LR White

RF–FS:

Rapid freezing–freeze substitution

RT:

Room temperature

RTS:

Rapid Transfer System

UA:

Uranyl acetate

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Acknowledgments

The author wishes to thank all those friends and collaborators who have provided materials used for these studies. Special thanks go to Richard I. Webb at the Microscopy and Microanalysis Centre, University of Queensland, Australia for helpful discussions and suggestions throughout the development of all the new rapid procedures reported here.

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The author declares that there is no conflict of interest.

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  1. Electron Microscope Laboratory, University of California, 26 Giannini Hall, Berkeley, CA, 94720, USA

    Kent L. McDonald

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McDonald, K.L. Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material. Protoplasma 251, 429–448 (2014). https://doi.org/10.1007/s00709-013-0575-y

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