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A Negative Stain for Electron Microscopic Tomography

Published online by Cambridge University Press:  27 February 2012

Andrea Fera ,
Jane E. Farrington ,
Joshua Zimmerberg  and
Thomas S. Reese
Andrea Fera*
Affiliation:
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA
Jane E. Farrington
Affiliation:
Laboratory of Cellular Molecular Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA
Joshua Zimmerberg
Affiliation:
Laboratory of Cellular Molecular Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA
Thomas S. Reese
Affiliation:
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA
*
Corresponding author. E-mail: [email protected]
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Abstract

While negative staining can provide detailed, two-dimensional images of biological structures, the potential of combining tomography with negative staining to provide three-dimensional views has yet to be fully realized. Basic requirements of a negative stain for tomography are that the density and atomic number of the stain are optimal, and that the stain does not degrade or rearrange with the intensive electron dose (∼106 e/nm2) needed to collect a full set of tomographic images. A commercially available, tungsten-based stain appears to satisfy these prerequisites. Comparison of the surface structure of negatively stained influenza A virus with previous structural results served to evaluate this negative stain. The combination of many projections of the same structure yielded detailed images of single proteins on the viral surface. Corresponding surface renderings are a good fit to images of the viral surface derived from cryomicroscopy as well as to the shapes of crystallized surface proteins. Negative stain tomography with the appropriate stain yields detailed images of individual molecules in their normal setting on the surface of the influenza A virus.

Keywords

tomographyinfluenza virusnegative stainingback projection methodindividual molecule imagingnative state imaging
Type
Biological and Biomedical Applications
Information
Microscopy and Microanalysis , Volume 18 , Issue 2 , April 2012 , pp. 331 - 335
Copyright
Copyright © Microscopy Society of America 2012

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