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.
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.
1Center 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.
Eric A
EA
Bushong
1Center 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
DD
Johnson
1Center 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
KY
Kim
2Carl Zeiss X-ray Microscopy Inc.,4385 Hopyard Rd #100,Pleasanton,CA 94588,USA.
Masako
M
Terada
3Salk Institute for Biological Sciences,10010 N Torrey Pines Rd,La Jolla,CA 92037,USA.
Megumi
M
Hatori
1Center 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.
Steven T
ST
Peltier
3Salk Institute for Biological Sciences,10010 N Torrey Pines Rd,La Jolla,CA 92037,USA.
Satchidananda
S
Panda
2Carl Zeiss X-ray Microscopy Inc.,4385 Hopyard Rd #100,Pleasanton,CA 94588,USA.
Arno
A
Merkle
1Center 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.
Mark H
MH
Ellisman
D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D021621:Imaging, Three-Dimensional; D051379:Mice; D008855:Microscopy, Electron, Scanning / Q000295:instrumentation / Q000379:methods*
upconverting nanoparticles