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Correlative cryogenic light and electron tomography of eukaryotic cells

Nature Reviews Methods Primers volume 5, Article number: 77 (2025) Cite this article

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Abstract

The importance of cryogenic electron tomography (cryo-ET), particularly cryogenic cellular tomography, in uncovering the complexity of eukaryotic cell systems is only beginning to be fully recognized. As the only structural technique capable of generating detailed, three-dimensional visualizations of cellular architecture in situ at sub-nanometre resolution, cryo-ET offers unmatched insights into cell biology and disease mechanisms. Integrating cryo-ET with light microscopy techniques allows researchers to localize specific regions of interest, then directly correlate them with high-resolution structural data, enabling the investigation of cellular components and dynamic processes with a level of precision previously unattainable. Despite its transformative potential, mastering integrative light microscopy techniques and cryo-ET workflows remains highly challenging. These techniques require specialized equipment, extensive hands-on experience and a deep understanding of practical nuances, many of which are acquired only through practice. In this Primer, we highlight key considerations, tips and common pitfalls to help guide newcomers to these approaches as they navigate how to begin and what challenges to anticipate.

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Fig. 1: A combined cryogenic correlative light and electron microscopy and cryogenic electron tomography workflow for adherent eukaryotic cells.
Fig. 2: Cryogenic electron tomography grids and grid tracking.
Fig. 3: Cryogenic electron microscopy substrates and maskless micropatterning do not compromise plasma membrane integrity.
Fig. 4: Cryogenic correlative light and electron microscopy tracking transport vesicles.
Fig. 5: Lamella targeting using cryo-confocal light microscopy.
Fig. 6: Post-milling fluorescence detection of NLR family pyrin domain-containing 3-activated apoptosis-associated speck-like protein complexes.
Fig. 7: Targeting of glutamatergic synapses with post-milling fluorescence.

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Acknowledgements

The authors thank M. Lemos and A. Bezault (Institut Pasteur) for their contributions to parts of the data presented here. D.H. acknowledges funding from the US Army Research Office under contract W911NF-19-D-0001 for the Institute for Collaborative Biotechnologies award. C.S. thanks the ANR-FRANCE (French National Research Agency) for its financial support, project no. ANR-22-CPJ2-0040-01. The authors acknowledge funding from the Institut Pasteur and the CNRS (D.H. and N.V.), and the NanoImaging Core (NCF) at Institut Pasteur for the provision of the equipment (Vitrobot, and cryo-electron microscopes). NanoImaging Core was created with the help of a grant from the French Government’s Investissements d’Avenir program (EQUIPEX CACSICE - Centre d’analyse de systèmes complexes dans les environements complexes, ANR-11-EQPX-0008).

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Authors and Affiliations

  1. Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France

    Cécile Sauvanet

  2. Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, CA, USA

    Peter Van Blerkom, Ahmet Hatipoglu & Dorit Hanein

  3. Department of Bioengineering, University of California Santa Barbara, Santa Barbara, CA, USA

    Niels Volkmann & Dorit Hanein

  4. Department of Electrical and Computer Engineering, University of California Santa Barbara, Santa Barbara, CA, USA

    Niels Volkmann

  5. Interdisciplinary Program for Quantitative Biosciences, University of California Santa Barbara, Santa Barbara, CA, USA

    Niels Volkmann

Authors
  1. Cécile Sauvanet
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  2. Peter Van Blerkom
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  3. Ahmet Hatipoglu
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  4. Niels Volkmann
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  5. Dorit Hanein
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Contributions

Introduction (C.S., N.V. and D.H.); Experimentation (C.S., P.V.B., A.H., N.V., and D.H.); Results (C.S., P.V.B., A.H., N.V. and D.H.); Applications (C.S., P.V.B., A.H., N.V. and D.H.); Reproducibility and data deposition (C.S., N.V. and D.H.); Limitations and optimization (C.S., N.V. and D.H.); Outlook (C.S., N.V. and D.H.).

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Correspondence to Dorit Hanein.

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Nature Reviews Methods Primers thanks Peter Dahlberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Bioimage Archive: https://www.ebi.ac.uk/bioimage-archive

EMDB: https://www.ebi.ac.uk/emdb

EMPIAR: https://www.ebi.ac.uk/empiar

Protein Data Bank (PDB): https://www.rcsb.org/

Supplementary information

Glossary

Correlative light and electron microscopy

(CLEM). Workflows that combine or integrate light and electron microscopy to precisely localize and correlate dynamic cellular events with structural snapshots.

Cryogenic focused ion beam milling

(Cryo-FIB milling). Process implemented to prepare lamellae at specific locations by ablating excess cellular material from the top and bottom of the sample.

Devitrification

Transition of water from the vitreous (amorphous) state to crystalline ice or liquid water.

Grids

Electron microscope supports made up of 3.05 mm-diameter circles, each consisting of a metal mesh coated with a thin film onto which samples are deposited for imaging.

Holey carbon film

Film support made from carbon that contains perforations of defined size and spacing.

Vitrification

Process of rapidly freezing water into its vitreous (amorphous) state, thereby preventing the formation of ice crystals.

Waffle freezing

Cryogenic electron tomography freezing method that consists of using a high-pressure-freezing machine and allowing to freeze thicker samples for subsequent cryogenic electron tomography experiments.

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Sauvanet, C., Van Blerkom, P., Hatipoglu, A. et al. Correlative cryogenic light and electron tomography of eukaryotic cells. Nat Rev Methods Primers 5, 77 (2025). https://doi.org/10.1038/s43586-025-00447-2

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