Brief Communication | Published:

Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography

Nature Methods volume 11, pages 737739 (2014) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Q. Rev. Biophys. 45, 27–56 (2012).

  2. 2.

    , , & Nat. Rev. Mol. Cell Biol. 7, 225–230 (2006).

  3. 3.

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

  4. 4.

    , & Curr. Opin. Biotechnol. 20, 83–89 (2009).

  5. 5.

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

  6. 6.

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

  7. 7.

    , , & in Methods Cell Biol. Vol. 96 (ed. Müller-Reichert, T.) Ch. 2, 21–45 (Academic Press, 2010).

  8. 8.

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

  9. 9.

    , & Nat. Methods 3, 793–795 (2006).

  10. 10.

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

  11. 11.

    , & Biophys. J. 91, 4258–4272 (2006).

  12. 12.

    , , , & J. Microsc. 227, 98–109 (2007).

  13. 13.

    , , , & Nat. Methods 9, 480–482 (2012).

  14. 14.

    & J. Microsc. 124, 3–4 (1981).

  15. 15.

    et al. Nature 475, 343–347 (2011).

  16. 16.

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

  17. 17.

    , , , & Nature 483, 182–186 (2012).

  18. 18.

    , & FEMS Microbiol. Rev. 34, 89–106 (2010).

  19. 19.

    , , , & EMBO J. 28, 315–325 (2009).

  20. 20.

    & Science 337, 815 (2012).

  21. 21.

    , , & Genes Dev. 10, 740–754 (1996).

  22. 22.

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

  23. 23.

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

  24. 24.

    & Proc. Natl. Acad. Sci. USA 90, 3378–3382 (1993).

  25. 25.

    , & Proc. Natl. Acad. Sci. USA 97, 9098–9103 (2000).

  26. 26.

    , , & Annu. Rev. Microbiol. 66, 453–472 (2012).

  27. 27.

    , , & Nat. Methods 7, 418–419 (2010).

  28. 28.

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

  29. 29.

    , & Biophys. J. 82, 2775–2783 (2002).

  30. 30.

    et al. J. Struct. Biol. 157, 138–147 (2007).

  31. 31.

    , & J. Struct. Biol. 116, 71–76 (1996).

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

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. Search for Yi-Wei Chang in:

  2. Search for Songye Chen in:

  3. Search for Elitza I Tocheva in:

  4. Search for Anke Treuner-Lange in:

  5. Search for Stephanie Löbach in:

  6. Search for Lotte Søgaard-Andersen in:

  7. Search for Grant J Jensen in:

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.

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.

Corresponding author

Correspondence to Grant J Jensen.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Tables 1–3

Videos

  1. 1.

    Demonstration of correlated cryo-PALM-CET

    Identification of T6SS structures in extended and contracted conformations in M. xanthus by correlated cryo-PALM-CET

  2. 2.

    Demonstration of correlated cryo-PALM-CET

    Identification of early assembly stage of T6SS structure in M. xanthus by correlated cryo-PALM-CET

About this article

Publication history

Received

Accepted

Published

DOI

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

Further reading