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Resources for the Study of Cellular Structure by High Voltage Electron Tomography, Serial Thin Sectioning, Specific Labeling, and Image Analysis

Published online by Cambridge University Press:  02 July 2020

David Mastronarde ,
James Kremer ,
Eileen O’Toole ,
Mary Morphew ,
Mark Ladinsky  and
Richard McIntosh
David Mastronarde
Affiliation:
Laboratory for 3-D Fine Structure, Dept. of MCD Biology, University of Colorado, Boulder, CO80309-0347
James Kremer
Affiliation:
Laboratory for 3-D Fine Structure, Dept. of MCD Biology, University of Colorado, Boulder, CO80309-0347
Eileen O’Toole
Affiliation:
Laboratory for 3-D Fine Structure, Dept. of MCD Biology, University of Colorado, Boulder, CO80309-0347
Mary Morphew
Affiliation:
Laboratory for 3-D Fine Structure, Dept. of MCD Biology, University of Colorado, Boulder, CO80309-0347
Mark Ladinsky
Affiliation:
Laboratory for 3-D Fine Structure, Dept. of MCD Biology, University of Colorado, Boulder, CO80309-0347
Richard McIntosh
Affiliation:
Laboratory for 3-D Fine Structure, Dept. of MCD Biology, University of Colorado, Boulder, CO80309-0347
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Extract

We are working to improve methods for the study of cellular fine structure. Our approach is to advance each of the key steps in the preparation of specimens for EM: high quality fixation that will preserve both structure and antigenicity; methods for specific labeling; efficient acquisition of 3-D electron microscopic data; and software for 3-D reconstruction and display.

Our work on high quality structure preservation has focused on methods for fast freezing and freeze substitution. Both plunge freezing of specimens grown on coated gold grids and high pressure freezing of either cultured cells or tissue specimens have yielded well preserved material. These samples are suitable for freeze substitution fixation with either anhydrous aldehydes in acetone at -90°C, for the preservation of antigens, or aldehydes, tannic acid, OsO4, and uranyl acetate for optimal preservation the structure.

We have used a JEOL JEM-1,000 high voltage microscope to image sections about 250nm thick, employing a goniometer stage to perform dual axis tomography for 3-D reconstruction with approximately isotropic resolution at ∼7nm.

Type
Shared Resources: Access to Critical Instrumentation
Information
Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 273 - 274
Copyright
Copyright © Microscopy Society of America 1997

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References

footnote

1. McDonald, K. and Morphew, M. (1993) Microscopy and Technique 24:465–473.CrossRefGoogle Scholar

2. McDonald, K., et al. (1996) Trends in Cell Biology, 24:465–473.Google Scholar

3. Mastronarde, D.M., et al. (1993) J. Cell Biol. 723:1475–1489.CrossRefGoogle Scholar

4. Kremer, J.R., et al. (1996) J. Struct. Biol. 116:71–76.CrossRefGoogle Scholar

5. This work was supported in part by RR 00592 from the NIH to JRM.Google Scholar