Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Gan, L. & Jensen, G.J. Q. Rev. Biophys. 45, 27–56 (2012).

    Article  CAS  Google Scholar 

  2. Nickell, S., Kofler, C., Leis, A.P. & Baumeister, W. Nat. Rev. Mol. Cell Biol. 7, 225–230 (2006).

    Article  CAS  Google Scholar 

  3. Briegel, A. et al. in Methods in Enzymology Vol. 481 (ed. Jensen, G.J.) Ch. 13, 317–341 (Academic Press, 2010).

  4. Plitzko, J.M., Rigort, A. & Leis, A. Curr. Opin. Biotechnol. 20, 83–89 (2009).

    Article  CAS  Google Scholar 

  5. Schlimpert, S. et al. Cell 151, 1270–1282 (2012).

    Article  CAS  Google Scholar 

  6. Patla, I. et al. Nat. Cell Biol. 12, 909–915 (2010).

    Article  CAS  Google Scholar 

  7. Pilhofer, M., Ladinsky, M.S., McDowall, A.W. & Jensen, G.J. in Methods Cell Biol. Vol. 96 (ed. Müller-Reichert, T.) Ch. 2, 21–45 (Academic Press, 2010).

  8. Pilhofer, M. et al. Environ. Microbiol. 16, 417–429 (2014).

    Article  CAS  Google Scholar 

  9. Rust, M.J., Bates, M. & Zhuang, X. Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  10. Betzig, E. et al. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  11. Hess, S.T., Girirajan, T.P.K. & Mason, M.D. Biophys. J. 91, 4258–4272 (2006).

    Article  CAS  Google Scholar 

  12. Schwartz, C.L., Sarbash, V.I., Ataullakhanov, F.I., McIntosh, J.R. & Nicastro, D. J. Microsc. 227, 98–109 (2007).

    Article  Google Scholar 

  13. Landgraf, D., Okumus, B., Chien, P., Baker, T.A. & Paulsson, J. Nat. Methods 9, 480–482 (2012).

    Article  CAS  Google Scholar 

  14. Dubochet, J. & McDowall, A.W. J. Microsc. 124, 3–4 (1981).

    Article  Google Scholar 

  15. Russell, A.B. et al. Nature 475, 343–347 (2011).

    Article  CAS  Google Scholar 

  16. Pukatzki, S. et al. Proc. Natl. Acad. Sci. USA 103, 1528–1533 (2006).

    Article  CAS  Google Scholar 

  17. Basler, M., Pilhofer, M., Henderson, G.P., Jensen, G.J. & Mekalanos, J.J. Nature 483, 182–186 (2012).

    Article  CAS  Google Scholar 

  18. Konovalova, A., Petters, T. & Søgaard-Andersen, L. FEMS Microbiol. Rev. 34, 89–106 (2010).

    Article  CAS  Google Scholar 

  19. Bönemann, G., Pietrosiuk, A., Diemand, A., Zentgraf, H. & Mogk, A. EMBO J. 28, 315–325 (2009).

    Article  Google Scholar 

  20. Basler, M. & Mekalanos, J.J. Science 337, 815 (2012).

    Article  CAS  Google Scholar 

  21. Søgaard-Andersen, L., Slack, F.J., Kimsey, H. & Kaiser, D. Genes Dev. 10, 740–754 (1996).

    Article  Google Scholar 

  22. Kaiser, D. Proc. Natl. Acad. Sci. USA 76, 5952–5956 (1979).

    Article  CAS  Google Scholar 

  23. Shi, X. et al. J. Bacteriol. 190, 613–624 (2008).

    Article  CAS  Google Scholar 

  24. Shi, W. & Zusman, D.R. Proc. Natl. Acad. Sci. USA 90, 3378–3382 (1993).

    Article  CAS  Google Scholar 

  25. Julien, B., Kaiser, A.D. & Garza, A. Proc. Natl. Acad. Sci. USA 97, 9098–9103 (2000).

    Article  CAS  Google Scholar 

  26. Silverman, J.M., Brunet, Y.R., Cascales, E. & Mougous, J.D. Annu. Rev. Microbiol. 66, 453–472 (2012).

    Article  CAS  Google Scholar 

  27. Preibisch, S., Saalfeld, S., Schindelin, J. & Tomancak, P. Nat. Methods 7, 418–419 (2010).

    Article  CAS  Google Scholar 

  28. Wolter, S. et al. Nat. Methods 9, 1040–1041 (2012).

    Article  CAS  Google Scholar 

  29. Thompson, R.E., Larson, D.R. & Webb, W.W. Biophys. J. 82, 2775–2783 (2002).

    Article  CAS  Google Scholar 

  30. Zheng, S.Q. et al. J. Struct. Biol. 157, 138–147 (2007).

    Article  CAS  Google Scholar 

  31. Kremer, J.R., Mastronarde, D.N. & McIntosh, J.R. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  Google Scholar 

Download references

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.

Author information

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

Authors
  1. Yi-Wei Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Songye Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Elitza I Tocheva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anke Treuner-Lange
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stephanie Löbach
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lotte Søgaard-Andersen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Grant J Jensen
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Ethics declarations

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)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2961

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology