DOI: 10.1111/jmi.12939. Advanced cryo‐tomography workflow developments – correlative microscopy, milling automation and cryo‐lift‐out

Advanced cryo‐tomography workflow developments – correlative microscopy, milling automation and cryo‐lift‐out

10.1111/jmi.12939

Crossref journal-article

Wiley

Journal of Microscopy (311)

Abstract

SummaryCryo‐electron tomography (cryo‐ET) is a groundbreaking technology for 3D visualisation and analysis of biomolecules in the context of cellular structures. It allows structural investigations of single proteins as well as their spatial arrangements within the cell. Cryo‐tomograms provide a snapshot of the complex, heterogeneous and transient subcellular environment. Due to the excellent structure preservation in amorphous ice, it is possible to study interactions and spatial relationships of proteins in their native state without interference caused by chemical fixatives or contrasting agents. With the introduction of focused ion beam (FIB) technology, the preparation of cellular samples for electron tomography has become much easier and faster. The latest generation of integrated FIB and scanning electron microscopy (SEM) instruments (dual beam microscopes), specifically designed for cryo‐applications, provides advances in automation, imaging and the preparation of high‐pressure frozen bulk samples using cryo‐lift‐out technology. In addition, correlative cryo‐fluorescence microscopy provides cellular targeting information through integrated software and hardware interfaces. The rapid advances, based on the combination of correlative cryo‐microscopy, cryo‐FIB and cryo‐ET, have already led to a wealth of new insights into cellular processes and provided new 3D image data of the cell. Here we introduce our recent developments within the cryo‐tomography workflow, and we discuss the challenges that lie ahead.Lay DescriptionThis article describes our recent developments for the cryo‐electron tomography (cryo‐ET) workflow. Cryo‐ET offers superior structural preservation and provides 3D snapshots of the interior of vitrified cells at molecular resolution. Before a cellular sample can be imaged by cryo‐ET, it must be made accessible for transmission electron microscopy. This is achieved by preparing a 200–300 nm thin cryo‐lamella from the cellular sample using a cryo‐focused ion beam (cryo‐FIB) microscope. Cryo‐correlative light and electron microscopy (cryo‐CLEM) is used within the workflow to guide the cryo‐lamella preparation to the cellular areas of interest. We cover a basic introduction of the cryo‐ET workflow and show new developments for cryo‐CLEM, which facilitate the connection between the cryo‐light microscope and the cryo‐FIB. Next, we present our progress in cryo‐FIB software automation to streamline cryo‐lamella preparation. In the final section we demonstrate how the cryo‐FIB can be used for 3D imaging and how bulk‐frozen cellular samples (obtained by high‐pressure freezing) can be processed using the newly developed cryo‐lift‐out technology.

Bibliography

KUBA, J., MITCHELS, J., HOVORKA, M., ERDMANN, P., BERKA, L., KIRMSE, R., KÃNIG, J., DE BOCK, J., GOETZE, B., & RIGORT, A. (2020). Advanced cryoâtomography workflow developments â correlative microscopy, milling automation and cryoâliftâout. Journal of Microscopy, 281(2), 112â124. Portico.

Authors 10

JAKUB KUBA (first)
JOHN MITCHELS (additional)
MILOŠ HOVORKA (additional)
PHILIPP ERDMANN (additional)
LUKÁŠ BERKA (additional)
ROBERT KIRMSE (additional)
JULIA KÖNIG (additional)
JAN DE BOCK (additional)
BERNHARD GOETZE (additional)
ALEXANDER RIGORT (additional)

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Dates

Type	When
Created	5 years, 2 months ago (June 18, 2020, 4:11 a.m.)
Deposited	1 year, 11 months ago (Sept. 3, 2023, 9:30 a.m.)
Indexed	34 minutes ago (Aug. 21, 2025, 1:12 a.m.)
Issued	5 years, 1 month ago (July 2, 2020)
Published	5 years, 1 month ago (July 2, 2020)
Published Online	5 years, 1 month ago (July 2, 2020)
Published Print	4 years, 6 months ago (Feb. 1, 2021)

Funders 1

Ministerstvo Školství, Mládeže a Tělovýchovy 10.13039/501100001823
Region: Europe
gov (National government)
Labels4
1. The Ministry of Education, Youth and Sports
2. MŠMT
3. MŠMT
4. MEYS
Awards1
1. LM2015062

BibTeX

@article{KUBA_2020, title={Advanced cryo‐tomography workflow developments – correlative microscopy, milling automation and cryo‐lift‐out}, volume={281}, ISSN={1365-2818}, url={http://dx.doi.org/10.1111/jmi.12939}, DOI={10.1111/jmi.12939}, number={2}, journal={Journal of Microscopy}, publisher={Wiley}, author={KUBA, JAKUB and MITCHELS, JOHN and HOVORKA, MILOŠ and ERDMANN, PHILIPP and BERKA, LUKÁŠ and KIRMSE, ROBERT and KÖNIG, JULIA and DE BOCK, JAN and GOETZE, BERNHARD and RIGORT, ALEXANDER}, year={2020}, month=jul, pages={112–124} }

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