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.

  • Article
  • Published:

Protein localization in electron micrographs using fluorescence nanoscopy

Nature Methods volume 8, pages 80–84 (2011)Cite this article

Subjects

Abstract

A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection protein in electron micrographs.

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: Correlative fluorescence and electron microscopy using histone H2B fusion proteins.
Figure 2: Correlative fluorescence and electron microscopy using TOM20 fusion proteins.
Figure 3: Correlative fluorescence and electron microscopy using α-liprin fusion proteins.

Similar content being viewed by others

References

  1. Cox, G. & Sheppard, C.J. Practical limits of resolution in confocal and non-linear microscopy. Microsc. Res. Tech. 63, 18–22 (2004).

    Article  Google Scholar 

  2. Hell, S.W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    Article  CAS  Google Scholar 

  3. Hell, S.W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

    Article  CAS  Google Scholar 

  4. Klar, T.A., Jakobs, S., Dyba, M., Egner, A. & Hell, S.W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).

    Article  CAS  Google Scholar 

  5. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  6. Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  7. Hess, S.T., Girirajan, T.P.K. & Mason, M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    Article  CAS  Google Scholar 

  8. Roth, J., Bendayan, M., Carlemalm, E., Villiger, W. & Garavito, M. Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J. Histochem. Cytochem. 29, 663–671 (1981).

    Article  CAS  Google Scholar 

  9. Rostaing, P., Weimer, R.M., Jorgensen, E.M., Triller, A. & Bessereau, J. Preservation of immunoreactivity and fine structure of adult C. elegans tissues using high-pressure freezing. J. Histochem. Cytochem. 52, 1–12 (2004).

    Article  CAS  Google Scholar 

  10. Morphew, M.K. 3D immunolocalization with plastic sections. Methods Cell Biol. 79, 493–513 (2007).

    Article  CAS  Google Scholar 

  11. Murphy, R.M. et al. Size and structure of antigen-antibody complexes. Electron microscopy and light scattering studies. Biophys. J. 54, 45–56 (1988).

    Article  CAS  Google Scholar 

  12. Sims, P.A. & Hardin, J.D. Fluorescence-integrated transmission electron microscopy images: integrating fluorescence microscopy with transmission electron microscopy. Methods Mol. Biol. 369, 291–308 (2007).

    Article  CAS  Google Scholar 

  13. Micheva, K. & Smith, S. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55, 25–36 (2007).

    Article  CAS  Google Scholar 

  14. Tsien, R. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  15. Mello, C.C., Kramer, J.M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970 (1991).

    Article  CAS  Google Scholar 

  16. Frøkjaer-Jensen, C. et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 40, 1375–1383 (2008).

    Article  Google Scholar 

  17. Yeh, E., Kawano, T., Weimer, R.M., Bessereau, J. & Zhen, M. Identification of genes involved in synaptogenesis using a fluorescent active zone marker in Caenorhabditis elegans. J. Neurosci. 25, 3833–3841 (2005).

    Article  CAS  Google Scholar 

  18. Riemersma, J.C. Osmium tetroxide fixation of lipids for electron microscopy. A possible reaction mechanism. Biochim. Biophys. Acta 152, 718–727 (1968).

    Article  CAS  Google Scholar 

  19. Clancy, B. & Cauller, L.J. Reduction of background autofluorescence in brain sections following immersion in sodium borohydride. J. Neurosci. Methods 83, 97–102 (1998).

    Article  CAS  Google Scholar 

  20. Newman, G.R. & Hobot, J.A. Resin Microscopy and On-Section Immuno-cytochemistry (Springer-Verlag, Berlin, 1993).

  21. Thompson, R. Precise nanometer localization snalysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    Article  CAS  Google Scholar 

  22. Yguerabide, J. & Yguerabide, E.E. Light-scattering submicroscopic particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications: II. Experimental characterization. Anal. Biochem. 262, 157–176 (1998).

    Article  CAS  Google Scholar 

  23. Goldstein, J. et al. Scanning Electron Microscopy and X-Ray Microanalysis (Springer Science and Business Media, LLS, New York, 2003).

  24. Hell, S.W., Lindek, S., Cremer, C. & Stelzer, E.H.K. Measurement of the 4Pi-confocal point spread function proves 75 nm axial resolution. Appl. Phys. Lett. 64, 1335–1337 (1994).

    Article  Google Scholar 

  25. Schmidt, R. et al. Mitochondrial cristae revealed with focused light. Nano Lett. 9, 2508–2510 (2009).

    Article  CAS  Google Scholar 

  26. Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl. Acad. Sci. USA 106, 3125–3130 (2009).

    Article  CAS  Google Scholar 

  27. Punge, A. et al. 3D reconstruction of high-resolution STED microscope images. Microsc. Res. Tech. 71, 644–650 (2008).

    Article  Google Scholar 

  28. Kanaji, S., Iwahashi, J., Kida, Y., Sakaguchi, M. & Mihara, K. Characterization of the signal that directs Tom20 to the mitochondrial outer membrane. J. Cell Biol. 151, 277–288 (2000).

    Article  CAS  Google Scholar 

  29. Clark, S.G., Lu, X. & Horvitz, H.R. The Caenorhabditis elegans locus Lin-15, a negative regulator of a tyrosine kinase dignaling pathway, encodes two different proteins. Genetics 137, 987–997 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Weibull, C. & Christiansson, A. Extraction of proteins and membrane lipids during low temperature embedding of biological material for electron microscopy. J. Microsc. 142, 79–86 (1986).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank H. Hess and E. Betzig (Janelia Farm) for access to the PALM microscope for proof-of-principle experiments; R. Fetter for sharing protocols, reagents and encouragement; M. Davidson (Florida State University), G. Seydoux (Johns Hopkins Univeristy), S. Eimer (European Neuroscience Institute), R. Leube (Universität Aachen), K. Nehrke (University of Rochester), C. Frøkjær-Jensen (Utah), A. Ada-Nguema (Utah) and M. Hammarlund (Yale University) for DNA constructs. We thank Marine Biological Laboratory for equipment and funding for pilot experiments and Carl Zeiss Inc. for providing access to a beta version of the PAL-M. This research was supported by the US National Institutes of Health (NS034307), National Science Foundation (0920069) and Marine Biology Laboratory fellowship. (The Dart Neuroscience Scholars Program in Learning and Memory).

Author information

Authors and Affiliations

  1. Department of Biology and Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah, USA

    Shigeki Watanabe, Gunther Hollopeter, Robert John Hobson, M Wayne Davis & Erik M Jorgensen

  2. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Annedore Punge, Katrin I Willig & Stefan W Hell

Authors
  1. Shigeki Watanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Annedore Punge
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gunther Hollopeter
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Katrin I Willig
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Robert John Hobson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M Wayne Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Stefan W Hell
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Erik M Jorgensen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.W. and E.M.J. conceived and designed experiments. G.H., R.J.H. and M.W.D. provided strains and advice. S.W. optimized the methods, prepared the samples and performed PALM imaging. A.P. and K.I.W. performed STED imaging. S.W., S.W.H. and E.M.J. wrote the manuscript. S.W.H. and E.M.J. provided funding.

Corresponding author

Correspondence to Erik M Jorgensen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 1, Supplementary Notes 1–5 (PDF 5940 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Watanabe, S., Punge, A., Hollopeter, G. et al. Protein localization in electron micrographs using fluorescence nanoscopy. Nat Methods 8, 80–84 (2011). https://doi.org/10.1038/nmeth.1537

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1537

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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