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X-Ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-Face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

Published online by Cambridge University Press:  13 November 2014

Eric A. Bushong ,
Donald D. Johnson Jr. ,
Keun-Young Kim ,
Masako Terada ,
Megumi Hatori ,
Steven T. Peltier ,
Satchidananda Panda ,
Arno Merkle  and
Mark H. Ellisman
Eric A. Bushong*
Affiliation:
Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
Donald D. Johnson Jr.
Affiliation:
Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
Keun-Young Kim
Affiliation:
Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
Masako Terada
Affiliation:
Carl Zeiss X-ray Microscopy Inc., 4385 Hopyard Rd #100, Pleasanton, CA 94588, USA
Megumi Hatori
Affiliation:
Salk Institute for Biological Sciences, 10010 N Torrey Pines Rd, La Jolla, CA 92037, USA
Steven T. Peltier
Affiliation:
Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
Satchidananda Panda
Affiliation:
Salk Institute for Biological Sciences, 10010 N Torrey Pines Rd, La Jolla, CA 92037, USA
Arno Merkle
Affiliation:
Carl Zeiss X-ray Microscopy Inc., 4385 Hopyard Rd #100, Pleasanton, CA 94588, USA
Mark H. Ellisman
Affiliation:
Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA Department of Neurosciences, University of California at San Diego School of Medicine, San Diego, La Jolla, CA 92093, USA
*
*Corresponding author.[email protected]
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Abstract

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.

Keywords

X-ray microscopymicrocomputed tomographyserial block-face scanning electron microscopycorrelative microscopyupconverting nanoparticles
Type
Biological and Biomaterials Applications
Information
Microscopy and Microanalysis , Volume 21 , Issue 1 , February 2015 , pp. 231 - 238
Copyright
© Microscopy Society of America 2014 

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Footnotes

†

Current address: Keio University, School of Medicine, 35 Shinanomachi Shinjyuku-ku, Tokyo 160-8582, Japan

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