Hostname: page-component-8448b6f56d-m8qmq Total loading time: 0 Render date: 2024-04-20T14:45:52.225Z Has data issue: false hasContentIssue false
  • English
  • Français

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.eric@ncmir.ucsd.edu
Get access

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 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

†

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

References

Arkill, K.P., Qvortrup, K., Starborg, T., Mantell, J.M., Knupp, C., Michel, C.C., Harper, S.J., Salmon, A.H., Squire, J.M., Bates, D.O. & Neal, C.R. (2014). Resolution of the three dimensional structure of components of the glomerular filtration barrier. BMC Nephrol 15(1), article no. 24.CrossRefGoogle ScholarPubMed
Auinger, S. & Small, J.V. (2008). Correlated light and electron microscopy of the cytoskeleton. Methods Cell Biol 88, 257–272.CrossRefGoogle ScholarPubMed
Burnett, T.L., McDonald, S.A., Gholinia, A., Geurts, R., Janus, M., Slater, T., Haigh, S.J., Ornek, C., Almuaili, F., Engelberg, D.L., Thompson, G.E. & Withers, P.J. (2014). Correlative tomography. Sci Rep 4, article no. 4711.CrossRefGoogle ScholarPubMed
Deerinck, T., Bushong, E., Lev-Ram, V., Shu, X., Tsien, R. & Ellisman, M. (2010). Enhancing serial block-face scanning electron microscopy to enable high resolution 3-D nanohistology of cells and tissues. Microsc Microanal 16(Suppl 2), 1138–1139.CrossRefGoogle Scholar
Denk, W. & Horstmann, H. (2004). Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2(11), e329.CrossRefGoogle ScholarPubMed
Hatori, M., Vollmers, C., Zarrinpar, A., DiTacchio, L., Bushong, E.A., Gill, S., Leblanc, M., Chaix, A., Joens, M., Fitzpatrick, J.A., Ellisman, M.H. & Panda, S. (2012). Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet. Cell Metab 15(6), 848–860.CrossRefGoogle ScholarPubMed
Helmstaedter, M. (2013). Cellular-resolution connectomics: Challenges of dense neural circuit reconstruction. Nat Methods 10(6), 501–507.CrossRefGoogle ScholarPubMed
Heymann, J.A., Shi, D., Kim, S., Bliss, D., Milne, J.L. & Subramaniam, S. (2009). 3D imaging of mammalian cells with ion-abrasion scanning electron microscopy. J Struct Biol 166(1), 1–7.CrossRefGoogle ScholarPubMed
Holcomb, P.S., Hoffpauir, B.K., Hoyson, M.C., Jackson, D.R., Deerinck, T.J., Marrs, G.S., Dehoff, M., Wu, J., Ellisman, M.H. & Spirou, G.A. (2013). Synaptic inputs compete during rapid formation of the calyx of Held: A new model system for neural development. J Neurosci 33(32), 12954–12969.CrossRefGoogle Scholar
Kleinfeld, D., Bharioke, A., Blinder, P., Bock, D.D., Briggman, K.L., Chklovskii, D.B., Denk, W., Helmstaedter, M., Kaufhold, J.P., Lee, W.C., Meyer, H.S., Micheva, K.D., Oberlaender, M., Prohaska, S., Reid, R.C., Smith, S.J., Takemura, S., Tsai, P.S. & Sakmann, B. (2011). Large-scale automated histology in the pursuit of connectomes. J Neurosci 31(45), 16125–16138.CrossRefGoogle ScholarPubMed
Knott, G., Marchman, H., Wall, D. & Lich, B. (2008). Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J Neurosci 28(12), 2959–2964.CrossRefGoogle ScholarPubMed
Leighton, S.B. (1981). SEM images of block faces, cut by a miniature microtome within the SEM—a technical note. Scan Electron Microsc Pt 2, 73–76.Google Scholar
Martell, J.D., Deerinck, T.J., Sancak, Y., Poulos, T.L., Mootha, V.K., Sosinsky, G.E., Ellisman, M.H. & Ting, A.Y. (2012). Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol 30(11), 1143–1148.CrossRefGoogle ScholarPubMed
McDonald, K., Schwarz, H., Muller-Reichert, T., Webb, R., Buser, C. & Morphew, M. (2010). “Tips and tricks” for high-pressure freezing of model systems. Methods Cell Biol 96, 671–693.CrossRefGoogle ScholarPubMed
Metscher, B.D. (2009). MicroCT for comparative morphology: Simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues. BMC Physiol 9, article no. 11.CrossRefGoogle ScholarPubMed
O’Brien, J.A. & Lummis, S.C. (2011). Nano-biolistics: A method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles. BMC Biotechnol 11, article no. 66.CrossRefGoogle ScholarPubMed
Peachey, L.D. (1958). Thin sections. I. A study of section thickness and physical distortion produced during microtomy. J Biophys Biochem Cytol 4(3), 233–242.CrossRefGoogle Scholar
Peddie, C.J. & Collinson, L.M. (2014). Exploring the third dimension: Volume electron microscopy comes of age. Micron 61C, 9–19.CrossRefGoogle Scholar
Pingel, J., Lu, Y., Starborg, T., Fredberg, U., Langberg, H., Nedergaard, A., Weis, M., Eyre, D., Kjaer, M. & Kadler, K.E. (2014). 3-D ultrastructure and collagen composition of healthy and overloaded human tendon: Evidence of tenocyte and matrix buckling. J Anat 224(5), 548–555.CrossRefGoogle ScholarPubMed
Puhka, M., Joensuu, M., Vihinen, H., Belevich, I. & Jokitalo, E. (2012). Progressive sheet-to-tubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells. Mol Biol Cell 23(13), 2424–2432.CrossRefGoogle ScholarPubMed
Sengle, G., Tufa, S.F., Sakai, L.Y., Zulliger, M.A. & Keene, D.R. (2013). A correlative method for imaging identical regions of samples by micro-CT, light microscopy, and electron microscopy: Imaging adipose tissue in a model system. J Histochem Cytochem 61(4), 263–271.CrossRefGoogle ScholarPubMed
Shu, X., Lev-Ram, V., Deerinck, T.J., Qi, Y., Ramko, E.B., Davidson, M.W., Jin, Y., Ellisman, M.H. & Tsien, R.Y. (2011). A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 9(4), e1001041.CrossRefGoogle ScholarPubMed
Stock, S.R. (2009). Microcomputed Tomography: Methodology and Applications. Boca Raton, FL: CRC Press.Google Scholar
West, J.B., Fu, Z., Deerinck, T.J., Mackey, M.R., Obayashi, J.T. & Ellisman, M.H. (2010). Structure-function studies of blood and air capillaries in chicken lung using 3D electron microscopy. Respir Physiol Neurobiol 170(2), 202–209.CrossRefGoogle ScholarPubMed
Wilke, S.A., Antonios, J.K., Bushong, E.A., Badkoobehi, A., Malek, E., Hwang, M., Terada, M., Ellisman, M.H. & Ghosh, A. (2013). Deconstructing complexity: Serial block-face electron microscopic analysis of the hippocampal mossy fiber synapse. J Neurosci 33(2), 507–522.CrossRefGoogle ScholarPubMed