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Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography

Nature Methods volume 11, pages 737–739 (2014)Cite this article

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Abstract

Cryo-electron tomography (CET) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated cryo-PALM (photoactivated localization microscopy)-CET method for localizing objects within cryo-tomograms to beyond the diffraction limit of the light microscope. Using cryo-PALM-CET, we identified multiple and new conformations of the dynamic type VI secretion system in the crowded interior of Myxococcus xanthus.

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Figure 1: Correlated cryo-PALM-CET used to visualize extended and contracted conformations of the T6SS sheath in M. xanthus.
Figure 2: Correlated cryo-PALM-CET identifies new T6SS structures.

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Acknowledgements

We thank A.W. McDowall, C. Oikonomou, A. Konovalova, L. Cai and T. Zhiyentayev for assistance and discussions. This work was supported in part by US National Institutes of Health grant R01 GM094800B to G.J.J., the Howard Hughes Medical Institute and the Max Planck Society.

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Authors and Affiliations

  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA

    Yi-Wei Chang, Songye Chen, Elitza I Tocheva & Grant J Jensen

  2. Howard Hughes Medical Institute, Pasadena, California, USA

    Yi-Wei Chang, Songye Chen & Grant J Jensen

  3. Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

    Anke Treuner-Lange, Stephanie Löbach & Lotte Søgaard-Andersen

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  1. Yi-Wei Chang
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Contributions

Y.-W.C. and G.J.J. conceived the cryo-PALM idea. Y.-W.C. and S.C. configured the optical system. Y.-W.C., S.C. and E.I.T. tested fluorophores for photoactivatability at low temperatures. Y.-W.C. improved stability of cryo-FLM stage, prepared samples, overcame laser-induced ice crystallization on the sample, acquired and analyzed cryo-PALM data and conducted correlated cryo-PALM-CET. A.T.-L., S.L. and L.S.-A. generated M. xanthus strains and conducted functional analyses. Y.-W.C. and G.J.J. wrote the paper with input from all authors.

Corresponding author

Correspondence to Grant J Jensen.

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Competing interests

Y.-W.C., S.C., E.I.T. and G.J.J. are affiliated with the California Institute of Technology, which has filed a patent application based on this work.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–3 (PDF 10761 kb)

Demonstration of correlated cryo-PALM-CET

Identification of T6SS structures in extended and contracted conformations in M. xanthus by correlated cryo-PALM-CET (MOV 26103 kb)

Demonstration of correlated cryo-PALM-CET

Identification of early assembly stage of T6SS structure in M. xanthus by correlated cryo-PALM-CET (MOV 25845 kb)

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Chang, YW., Chen, S., Tocheva, E. et al. Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nat Methods 11, 737–739 (2014). https://doi.org/10.1038/nmeth.2961

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