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Automated Montaging HVEM Tomography of Large Cellular Volumes

Published online by Cambridge University Press:  02 July 2020

D. N. Mastronarde ,
B. J. Marsh  and
M. Otegui
D. N. Mastronarde
Affiliation:
Boulder Laboratory for 3-Dimensional Fine Structure, University of Colorado, Boulder, CO, 80309
B. J. Marsh
Affiliation:
Boulder Laboratory for 3-Dimensional Fine Structure, University of Colorado, Boulder, CO, 80309
M. Otegui
Affiliation:
Dept. of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO, 80309
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Abstract

We have developed methods for generating tomographic reconstructions of large cellular volumes efficiently and at relatively high resolution. One key component of these methods is the ability to capture images from our high voltage electron microscope (HVEM) with a sensitive lenscoupled CCD camera. to overcome the limited resolution and field of view of this 1k × 1K camera, we have used the image shift capability of the HVEM to develop automatic montaging of images from an array of positions, with about 10% overlap between adjacent positions. We have also developed automatic repositioning and autofocusing after each tilt of the specimen, so that it is now possible to acquire single-frame or montaged images at a series of tilt angles, with no operator intervention. with these capabilities, up to 2 GB of image data can be acquired in a single session.

Tilt series are processed with the IMOD software package developed in our laboratory, which now runs on a PC under Linux, as well as on SGI computers. Several features are notable for working with large data sets.

Type
Electron Tomography: Recent Advances and Applications (Organized by M. Marko)
Information
Microscopy and Microanalysis , Volume 7 , Issue S2: Proceedings: Microscopy & Microanalysis 2001, Microscopy Society of America 59th Annual Meeting, Microbeam Analysis Society 35th Annual Meeting, Long Beach, California August 5-9, 2001 , August 2001 , pp. 90 - 91
Copyright
Copyright © Microscopy Society of America 2001

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References

1. This work was supported by NIH/NCRR grant #RR00592 to J.R. Mcintosh and NIH grant #GM59787 to L.A. Staehelin. B.J.M. is a JDRF Postdoctoral Fellow.Google Scholar