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Is Negative Staining Capable of High Resolution?

Published online by Cambridge University Press:  02 July 2020

William H. Massover
William H. Massover*
Affiliation:
Department of Anatomy and Cell Biology, UMDNJ - New Jersey Medical School, Newark, N.J.07103, USA
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Abstract

Everyone presently answers “no” to this question, since this methodology for specimen preparation and preservation commonly is believed to be inherently limited to 20-25Å (2.0-2.5nm) resolution. Achievement of resolution levels smaller than this figure have been published only rarely. This report presents experimental evidence from electron diffraction showing that negative staining can preserve periodic protein structure to the level of at least 4Å.

A suspension of bovine liver catalase crystals (orthorhombic: a= 69Å, b= 174Å, c= 206 Å [1]) is deposited on a hydrophilic carbon support and negatively stained using an on-grid protocol [2,3]. For low-dose electron diffraction with a JEOL 100CX transmission electron microscope (l00kV), the set-up for routine selected area diffraction is modified to keep the second condenser lens maximally overfocused; by a defocusing of the diffraction pattern, the grid can be surveyed and suitable thin single crystals centered in the diffraction aperture with only negligible irradiation.

Type
Diffraction Techniques in TEM and SEM
Information
Microscopy and Microanalysis , Volume 7 , Issue S2: Proceedings: Microscopy & Microanalysis 2001, Microscopy Society of America 59th Annual Meeting, Microbeam Analysis Society 35th Annual Meeting, Long Beach, California August 5-9, 2001 , August 2001 , pp. 360 - 361
Copyright
Copyright © Microscopy Society of America 2001

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References

1.Unwin, P. N. T.. J. Mol. Biol. 98 (1975) 235.CrossRefGoogle Scholar
2.Massover, W. H. and Marsh, P.. Ultramicroscopy 69 (1997) 139.CrossRefGoogle Scholar
3.Massover, w. H. and Marsh, P.. Ultramicroscopy 85 (2000) 107.CrossRefGoogle Scholar
This investigation was supported in part by research grant GM57948 from the National Institute of General Medical Sciences (NIH, USPHS, DHHS).Google Scholar