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Published June 2022 | Accepted Version + Submitted + Supplemental Material
Journal Article Open

Montage electron tomography of vitrified specimens

Abstract

Cryo-electron tomography provides detailed views of macromolecules in situ. However, imaging a large field of view to provide more cellular context requires reducing magnification during data collection, which in turn restricts the resolution. To circumvent this trade-off between field of view and resolution, we have developed a montage data collection scheme that uniformly distributes the dose throughout the specimen. In this approach, sets of slightly overlapping circular tiles are collected at high magnification and stitched to form a composite projection image at each tilt angle. These montage tilt-series are then reconstructed into massive tomograms with a small pixel size but a large field of view. For proof-of-principle, we applied this method to the thin edge of HeLa cells. Thon rings to better than 10 Å were detected in the montaged tilt-series, and diverse cellular features were observed in the resulting tomograms. These results indicate that the additional dose required by this technique is not prohibitive to performing structural analysis to intermediate resolution across a large field of view. We anticipate that montage tomography will prove particularly useful for lamellae, increase the likelihood of imaging rare cellular events, and facilitate visual proteomics.

Additional Information

© 2022 Elsevier Inc. Received 15 November 2021, Revised 25 March 2022, Accepted 20 April 2022, Available online 26 April 2022, Version of Record 6 May 2022. Data collection and analysis were respectively performed at the Beckman Institute Resource Center for Transmission Electron Microscopy and the Resnick High Performance Computing Center at Caltech. We thank Wei Zhao for preliminary samples, David Mastronarde for valuable discussions, and Tom Morrell for help with the Caltech Data Repository. A.P. is The Mark Foundation for Cancer Research Fellow of the Damon Runyon Cancer Research Foundation (DRG 2361-19). This work was supported by NIH grant AI127401 to G.J.J. Data and code availability: Raw data are available at the Caltech Data Repository (https://data.caltech.edu) under accession IDs 2096, 2099, and 2103. The processed tilt-series binned to 5.3 Å can be found in the Caltech Electron Tomography Database (https://etdb.caltech.edu/). The code developed for data collection and processing is available at https://github.com/apeck12/montage. CRediT authorship contribution statement: Ariana Peck: Methodology, Software, Writing – original draft, Writing – review & editing. Stephen D. Carter: Conceptualization, Methodology, Investigation. Huanghao Mai: Methodology, Software. Songye Chen: Software, Methodology, Investigation. Alister Burt: Formal analysis, Writing – review & editing. Grant J. Jensen: Conceptualization, Writing – original draft, Writing – review & editing, Funding acquisition, Supervision. Declaration of Competing Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Attached Files

Accepted Version - 1-s2.0-S1047847722000302-main-1.pdf

Accepted Version - nihms-1871136.pdf

Submitted - 2021.11.02.466666v1.full.pdf

Supplemental Material - 1-s2.0-S1047847722000302-mmc1.pdf

Supplemental Material - 1-s2.0-S1047847722000302-mmc2.mp4

Supplemental Material - 1-s2.0-S1047847722000302-mmc3.mp4

Supplemental Material - 1-s2.0-S1047847722000302-mmc4.mp4

Supplemental Material - 1-s2.0-S1047847722000302-mmc5.mp4

Supplemental Material - 1-s2.0-S1047847722000302-mmc6.mp4

Supplemental Material - 1-s2.0-S1047847722000302-mmc7.mp4

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Additional details

Created:
August 22, 2023
Modified:
December 22, 2023