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Automated Data Collection with a Tecnai 12 Electron Microscope for Applications in Structural Biology

Published online by Cambridge University Press:  02 July 2020

Peijun Zhang
Affiliation:
laboratories of Biochemistry National Institutes of Health, Bethesda, MD, 20817
Alexis Beatty
Affiliation:
laboratories of Biochemistry National Institutes of Health, Bethesda, MD, 20817
Jacqueline L.S. Milne
Affiliation:
Cell Biology , National Cancer Institute, National Institutes of Health, Bethesda, MD, 20817
Sriram Subramaniam
Affiliation:
laboratories of Biochemistry National Institutes of Health, Bethesda, MD, 20817

Abstract

In high-resolution biological electron microscopy, the speed of collection of large numbers of micrographs of the required quality is a rate-limiting step in the overall process of structure determination. Approaches to speed up data collection can be very useful especially in “single molecule” microscopy of large multi-protein and protein-nucleic acid complexes, where many thousands of individual molecular images need to be averaged to determine the threedimensional structure.

Here, we report the development of an automated low-dose image acquisition procedure on a Tecnai 12 electron microscope using the scripting functionality available on the microscope computer. The program that we have developed and implemented, AutoEM, is presently aimed at providing automation capabilities for the user familiar with normal operation of the microscope. However, the user’s participation is relatively minimal, and involves the following steps. After preparing a specimen grid and inserting it into the microscope as is normal for a cryo transfer session, the user simply downloads a Low-Dose alignment file that contains information on the relative beam shifts between the Search, Focus and Exposure modes that would normally be employed in a manual low-dose data collection experiment.

Type
Instrument Automation (Organized by W. Deruijter and C. Potter)
Copyright
Copyright © Microscopy Society of America 2001

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