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:

Nanoscale imaging of molecular positions and anisotropies

Nature Methods volume 5, pages 1027–1030 (2008)Cite this article

Abstract

Knowledge of the orientation of molecules within biological structures is crucial to understanding the mechanisms of cell function. We present a method to image simultaneously the positions and fluorescence anisotropies of large numbers of single molecules with nanometer lateral resolution within a sample. Based on a simple modification of fluorescence photoactivation localization microscopy (FPALM), polarization (P)-FPALM does not compromise speed or sensitivity. We show results for mouse fibroblasts expressing Dendra2-actin or Dendra2-hemagglutinin.

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: P-FPALM detection path and P-FPALM imaging of a fixed fibroblast expressing Dendra2-actin.
Figure 2: Effect of cytochalasin-D on structure and anisotropy of Dendra2-actin.

Similar content being viewed by others

References

  1. Hell, S.W. & Wichmann, J. Opt. Lett. 19, 780–782 (1994).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Hess, S.T. et al. Proc. Natl. Acad. Sci. USA 104, 17370–17375 (2007).

    Article  CAS  Google Scholar 

  6. Shroff, H., Galbraith, C.G., Galbraith, J.A. & Betzig, E. Nat. Methods 5, 417–423 (2008).

    Article  CAS  Google Scholar 

  7. Huang, B., Wang, W., Bates, M. & Zhuang, X. Science 319, 810–813 (2008).

    Article  CAS  Google Scholar 

  8. Juette, M.F. et al. Nat. Methods 5, 527–529 (2008).

    Article  CAS  Google Scholar 

  9. Betzig, E. & Chichester, R.J. Science 262, 1422–1425 (1993).

    Article  CAS  Google Scholar 

  10. Bartko, A.P. & Dickson, R.M. J. Phys. Chem. B 103, 11237–11241 (1999).

    Article  CAS  Google Scholar 

  11. Bohmer, M. & Enderlein, J. J. Opt. Soc. Am. B 20, 554–559 (2003).

    Article  CAS  Google Scholar 

  12. Harms, G.S., Cognet, L., Lommerse, P.H.M., Blab, G.A. & Schmidt, T. Biophys. J. 80, 2396–2408 (2001).

    Article  CAS  Google Scholar 

  13. Patterson, G.H. & Lippincott-Schwartz, J. Science 297, 1873–1877 (2002).

    Article  CAS  Google Scholar 

  14. Gurskaya, N.G. et al. Nat. Biotechnol. 24, 461–465 (2006).

    Article  CAS  Google Scholar 

  15. Fourkas, J.T. Opt. Lett. 26, 211–213 (2001).

    Article  CAS  Google Scholar 

  16. Lakowicz, J.R. Principles of Fluorescence Spectroscopy 3rd ed. (Springer Science, New York, 2006).

    Book  Google Scholar 

  17. Weber, G. J. Opt. Soc. Am. 46, 962–970 (1956).

    Article  CAS  Google Scholar 

  18. Hess, S.T., Sheets, E.D., Wagenknecht-Wiesner, A. & Heikal, A.A. Biophys. J. 85, 2566–2580 (2003).

    Article  CAS  Google Scholar 

  19. Jacobson, K., Mouritsen, O.G. & Anderson, R.G. Nat. Cell Biol. 9, 7–14 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Fang-Yen, P. Blank, J. Bewersdorf, J. Zimmerberg and M. Mason for useful discussions, G. Patterson (US National Institute of Child Health and Human Development) for providing the construct encoding the PA-GFP protein, J. Shim, J. Rochira and E. Allgeyer for laboratory assistance, A. McGinn, T. Tripp and P. Byard for professional services. This work was supported by grants K25-65459 from the US National Institute of Allergy and Infectious Diseases, CHE-0722759 from the National Science Foundation, start up funds from the University of Maine (S.T.H.), and by grants GM070358 and GM073913 from the National Institute of General Medical Sciences (V.V.V.).

Author information

Author notes

  1. Travis J Gould and Mudalige S Gunewardene: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physics and Astronomy and Institute for Molecular Biophysics, 5709 Bennett Hall, University of Maine, Orono, 04469, Maine, USA

    Travis J Gould, Mudalige S Gunewardene, Manasa V Gudheti & Samuel T Hess

  2. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, 10461, New York, USA

    Vladislav V Verkhusha

  3. Laboratory for Cellular and Molecular Biophysics, Program in Physical Biology, National Institute of Child Health and Human Development, 10 Center Drive, Bethesda, 20892, Maryland, USA

    Shu-Rong Yin

  4. Department of Biochemistry, Microbiology, and Molecular Biology, 5735 Hitchner Hall, University of Maine, Orono, 04469, Maine, USA

    Julie A Gosse

Authors
  1. Travis J Gould
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mudalige S Gunewardene
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Manasa V Gudheti
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vladislav V Verkhusha
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shu-Rong Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Julie A Gosse
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Samuel T Hess
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.J.G. and M.S.G. conceived the method, performed and analyzed experiments, wrote and edited the manuscript. M.V.G. performed experiments and edited the manuscript. V.V.V. and S.-R.Y. created genetic constructs and edited the manuscript. J.A.G. assisted with experiments and analysis, and edited the manuscript. S.T.H. conceived the method, performed and analyzed experiments, wrote and edited the manuscript.

Corresponding author

Correspondence to Samuel T Hess.

Ethics declarations

Competing interests

T.J.G., M.S.G., and S.T.H. have applied for a provisional patent on the instrumentation described in this manuscript.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–17, Supplementary Tables 1–2, Supplementary Methods (PDF 4109 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gould, T., Gunewardene, M., Gudheti, M. et al. Nanoscale imaging of molecular positions and anisotropies. Nat Methods 5, 1027–1030 (2008). https://doi.org/10.1038/nmeth.1271

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1271

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