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Automated Acquisition of Cryo Electron Micrographs Using Leginon

Published online by Cambridge University Press:  02 July 2020

B. Carragher
Affiliation:
Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana- Champaign, Urbana, IL61801
N. Jojic
Affiliation:
Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana- Champaign, Urbana, IL61801
R. A. Milligan
Affiliation:
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA92037
N. Kisseberth
Affiliation:
Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana- Champaign, Urbana, IL61801
J. Pulokas
Affiliation:
Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana- Champaign, Urbana, IL61801
C.S. Potter
Affiliation:
Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana- Champaign, Urbana, IL61801
A. Reilein
Affiliation:
Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana- Champaign, Urbana, IL61801

Extract

Molecular microscopy is one of the most important structural approaches in cell biological investigations and can provide insight into complex biological questions that no other technique can provide. Currently, the technique typically requires the acquisition of very large numbers of transmission electron micrographs from frozen hydrated specimens using low dose techniques. The field is constrained by manual data acquisition methods that are slow, labor-intensive and result in a very low percentage of suitable images. We have developed a system, called Leginon, for automatically acquiring images from a transmission electron microscope. Our first prototype of this system demonstrated that we could acquire 1000 high magnification images per day from negatively stained catalase crystals. We have now extended this system to acquire low dose images of specimens embedded in vitreous ice.

Methods: Specimens were prepared on Quantifoil grids using techniques which have been described previously. The Leginon system uses a Philips CM200 TEM and a Gatan MSC CCD camera and is controlled by the emScope software library. The overall acquisition protocol requires (i) obtaining a low magnification image [660x] of a grid square from a Quantifoil grid (fig. 1a); (ii) automatically identifying holes containing ice of suitable thickness; (iii) acquiring an intermediate magnification image [6600x] of the identified hole (fig. 1b); (iv) identifying features of interest within the hole; (v) focusing at high magnification [38,000x] and finally (vi) acquiring a high magnification image (fig 1c,d).

Type
Advances in Digital Imaging
Copyright
Copyright © Microscopy Society of America

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References

1.Potter, C.S.et al., Proc. Microscopy and Microanalysis (1998) 89.Google Scholar
2.Potter, C. S.et al., Submitted for publication.Google Scholar
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7.Pulokas, J.et al., Submitted to this conference.Google Scholar
8. Support is provided by the NSF (9730056) and the IBM Shared University Research program.Google Scholar

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