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Super-Thin Serial Sectioning for High-Resolution 3-D Reconstruction of Cellular Structures

Published online by Cambridge University Press:  02 July 2020

D. N. Mastronarde
Affiliation:
Boulder Laboratory for 3-D Fine Structure, Department of MCD Biology, University of Colorado, Boulder, CO, 80309
M. S. Ladinsky
Affiliation:
Boulder Laboratory for 3-D Fine Structure, Department of MCD Biology, University of Colorado, Boulder, CO, 80309
J. R. Mcintosh
Affiliation:
Boulder Laboratory for 3-D Fine Structure, Department of MCD Biology, University of Colorado, Boulder, CO, 80309
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Extract

We are developing techniques for obtaining the thinnest possible serial sections of biological specimens. Our goal is to produce not just an occasional very thin section but large numbers of 5-15 nm serial sections suitable for 3-D reconstruction. We have worked out conditions that allow us routinely to cut serial sections ˜15 nm thick, and have achieved ribbons of sections with an average thickness of only 11 nm.

We have worked with the Leica Ultracut S and Ultracut UCT microtomes, which have proven to be exceptionally stable instruments, capable of very regular advance between cutting strokes. The resulting low variability in section thickness is essential for serial sections thinner than 15 nm. However, these machines have an inherent forward drift that increases the thickness of sections cut soon after a block has been mounted and prepared for cutting. This drift is initially 50-100 nm/min, but decreases to only 10-20 nm/min if the microtome is allowed to cycle for 0.5—1.5 hours.

Type
Innovative Approaches to 3-D Structure/Function Determination for Cells and Organelles
Copyright
Copyright © Microscopy Society of America 1997

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References

1.DeGroot, D. M. G., Journal of Microscopy, 151(1988)23.Google Scholar
2.Mastronarde, D. N. et al., Journal of Cell Biology, 118(1992)1145.10.1083/jcb.118.5.1145CrossRefGoogle Scholar
3. This work was supported by NIH grant RR00592 to McIntosh, J. R..Google Scholar