Automated data collection in single particle electron microscopy - PubMed (original) (raw)

Review

Automated data collection in single particle electron microscopy

Yong Zi Tan et al. Microscopy (Oxf). 2016 Feb.

Abstract

Automated data collection is an integral part of modern workflows in single particle electron microscopy (EM) research. This review surveys the software packages available for automated single particle EM data collection. The degree of automation at each stage of data collection is evaluated, and the capabilities of the software packages are described. Finally, future trends in automation are discussed.

Keywords: automation; data collection; high throughput; single particle electron microscopy; software packages; target acquisition.

© The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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Figures

Fig. 1.

Fig. 1.

Negative stain electron micrographs of the same field at different defocus values. (a) is closest to focus, followed by (b) and (c) with sequentially higher defocus values. The corresponding Fourier transforms of each micrograph are shown (d–f). Defocus values were estimated using CTFFind3 [38]. Images had a pixel size of 1.83 Å and were taken with a 200 kV microscope with _C_s of 2.0 mm.

Fig. 2.

Fig. 2.

Comparison of non-astigmatic (a) and strongly astigmatic (b) micrographs. Their corresponding Fourier transforms are shown below (c and d). Images had a pixel size of 1.83 Å and were taken with a 200-kV microscope with _C_s of 2.0 mm.

Fig. 3.

Fig. 3.

Zemlin tableaus acquired with coma-free alignment (a) and without coma-free alignment (b). The calculated resolution limit (based on Eq. (2)) for the aberration seen in (b) and corrected in (a) is ∼0.55 nm, given a resolution limit cutoff at 45° phase error, using a 200 kV microscope with _C_s of 2.0 mm.

Fig. 4.

Fig. 4.

MSI procedure employed in data collection software for both cryo T20S proteasome (a) and negative stain 50S ribosome (b) samples. The white boxes indicate the area where an image is acquired at subsequent higher magnification and the white arrows point to a single particle of the respective proteins.

Fig. 5.

Fig. 5.

Variation of squares in a typical negative stain (a) and cryo-EM (b) grid. An example of a broken square (yellow), a square with too thick ice/stain (red) and a potentially good square (green) are shown in the respective colors above.

Fig. 6.

Fig. 6.

Electron micrographs of negative stain samples at 0° tilt (a) and −55° tilt (b).

Fig. 7.

Fig. 7.

Remote monitoring of data collection by LOI.

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