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High-Resolution Analysis of Rapidly Frozen Biological Specimens: Capabilities and Limitations

Published online by Cambridge University Press:  02 July 2020

S.B. Andrews ,
N.B. Pivovarova ,
J. Hongpaisan  and
R.D. Leapman
S.B. Andrews
Affiliation:
Laboratory of Neurobiology, NINDS OD, National Institutes of Health, Bethesda, MD20892.
N.B. Pivovarova
Affiliation:
Laboratory of Neurobiology, NINDS OD, National Institutes of Health, Bethesda, MD20892.
J. Hongpaisan
Affiliation:
Laboratory of Neurobiology, NINDS OD, National Institutes of Health, Bethesda, MD20892.
R.D. Leapman
Affiliation:
Bioengineering & Physical Science Program, OD, National Institutes of Health, Bethesda, MD20892.
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Extract

The past decade has seen major advances in the analytical capability and utility of both fixed beam and scanning beam electron microscopes. In particular, scanning transmission electron microscopy (STEM) and energy-filtering transmission microscopy (EFTEM) have benefited from the development of devices and techniques—including improved electron optics, sensitive solid-state detectors and new software for imaging and electron energy loss spectroscopy (EELS)—that optimize detection of weak spectroscopic signals arising from biological specimens while minimizing specimen damage. Here we discuss and illustrate some of these advances, especially in the context of structural imaging, detection limits and mapping techniques for the biologically important elements phosphorus and calcium. Analytical microscopy of biological tissues is absolutely dependent on cryotechniques. It is generally agreed that rapid freezing and subsequent low-temperature processing, e.g., cryosectioning or direct cryotransfer of frozen-hydrated specimens, is the most reliable way to preserve the native distribution and organization of biological structures. Equally important, however, as an adjunct to spectroscopic analysis is the use of established low-temperature, low-dose techniques for recording optimized images. By limiting beam exposure, low-dose methods greatly improve the quality of images from fragile, freeze-dried preparations. In this case, the quality and information content of, e.g., cryosections are virtually as good as conventional preparations (Fig 1).

Type
Cryotechniques, Immunocytochemistry, and Electron Microscopy I. Molecular Approach
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 412 - 413
Copyright
Copyright © Microscopy Society of America

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