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:

A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy

Nature Methods volume 8, pages 143–146 (2011)Cite this article

Subjects

Abstract

Time resolution of current single-molecule fluorescence techniques is limited to milliseconds because of dye blinking and bleaching. Here we introduce a photoprotection strategy that affords microsecond resolution by combining efficient triplet quenching by oxygen and Trolox with minimized bleaching via the oxygen radical scavenger cysteamine. Using this approach we resolved the single-molecule microsecond conformational fluctuations of two proteins: the two-state folder α-spectrin SH3 domain and the ultrafast downhill folder BBL.

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: Effect of photoprotectors on the photon throughput of B-DNA10 labeled with A488-A594 at 10-base-pair separation.
Figure 2: Microsecond-resolution free-diffusion single-molecule FRET experiments of α-spectrin SH3 domain labeled with A488-A594 on two cysteines introduced at the protein ends.
Figure 3: Microsecond resolution free diffusion single-molecule FRET of BBL.

Similar content being viewed by others

References

  1. Moerner, W.E. Proc. Natl. Acad. Sci. USA 104, 12596–12602 (2007).

    Article  CAS  Google Scholar 

  2. Roy, R., Hohng, S. & Ha, T. Nat. Methods 5, 507–516 (2008).

    Article  CAS  Google Scholar 

  3. Schuler, B. & Eaton, W.A. Curr. Opin. Struct. Biol. 18, 16–26 (2008).

    Article  CAS  Google Scholar 

  4. Gell, C., Brockwell, D. & Smith, A. Handbook of Single Molecule Fluorescence Spectroscopy. (Oxford University Press, 2006).

    Google Scholar 

  5. Kong, X.X., Nir, E., Hamadani, K. & Weiss, S. J. Am. Chem. Soc. 129, 4643–4654 (2007).

    Article  CAS  Google Scholar 

  6. Rasnik, I., McKinney, S.A. & Ha, T. Nat. Methods 3, 891–893 (2006).

    Article  CAS  Google Scholar 

  7. Turro, N.J. Modern Molecular Photochemistry. (University Science Books, 1991).

    Google Scholar 

  8. Dittrich, P.S. & Schwille, P. Appl. Phys. B 73, 829–837 (2001).

    Article  CAS  Google Scholar 

  9. Misik, V., Miyoshi, N. & Riesz, P. Free Radic. Biol. Med. 26, 961–967 (1999).

    Article  CAS  Google Scholar 

  10. Wozniak, A.K., Schroder, G.F., Grubmuller, H., Seidel, C.A. & Oesterhelt, F. Proc. Natl. Acad. Sci. USA 105, 18337–18342 (2008).

    Article  CAS  Google Scholar 

  11. Li, P., Oliva, F.Y., Naganathan, A.N. & Muñoz, V. Proc. Natl. Acad. Sci. USA 106, 103–108 (2009).

    Article  CAS  Google Scholar 

  12. Muñoz, V. Annu. Rev. Biophys. Biomol. Struct. 36, 395–412 (2007).

    Article  Google Scholar 

  13. Eaton, W.A. et al. Annu. Rev. Biophys. Biomol. Struc. 29, 327–359 (2000).

    Article  CAS  Google Scholar 

  14. Fawni, N.L., Douclef, M., Suh, J.Y. & Clore, M.G. Proc. Natl. Acad. Sci. USA 107, 1379–1384 (2010).

    Article  Google Scholar 

  15. Neuweiler, H., Johnson, C.M. & Fersht, A.R. Proc. Natl. Acad. Sci. USA 106, 18569–18574 (2009).

    Article  CAS  Google Scholar 

  16. Morgan, M.A., Okamoto, K., Kahn, J.D. & English, D.S. Biophys. J. 89, 2588–2596 (2005).

    Article  CAS  Google Scholar 

  17. Gopich, I.V. & Szabo, A. J. Phys. Chem. B 109, 17683–17688 (2005).

    Article  CAS  Google Scholar 

  18. McKinney, S.A., Joo, C. & Ha, T. Biophys. J. 91, 1941–1951 (2006).

    Article  CAS  Google Scholar 

  19. Lapidus, L.J., Steinbach, P.J., Eaton, W.A., Szabo, A. & Hofrichter, J. J. Phys. Chem. B 106, 11628–11640 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported in part by the Marie Curie excellence grant MEXT-CT-2006-042334 and grants BFU2008-03237 and CONSOLIDER CSD2009-00088 from the Spanish Ministry of Science and Innovation.

Author information

Author notes

  1. Jianwei Liu, Xiang Wang & Douglas S English

    Present address: Present addresses: Department of Pediatrics, Stanford University, Stanford, California, USA (J.L.), Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, USA (X.W.) and Department of Chemistry, Wichita State University, Wichita, Kansas, USA (D.S.E.).,

  2. Luis A Campos and Jianwei Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain

    Luis A Campos, Ravishankar Ramanathan & Victor Muñoz

  2. Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, USA

    Luis A Campos, Jianwei Liu, Xiang Wang, Douglas S English & Victor Muñoz

Authors
  1. Luis A Campos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jianwei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ravishankar Ramanathan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Douglas S English
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Victor Muñoz
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.A.C. and J.L. prepared samples, identified the oxygen radical scavengers, performed experiments and analyzed data. L.A.C. performed all the additional experiments requested by the reviewers. X.W. acquired and analyzed data. R.R. performed the stochastic simulations of downhill folding. D.S.E. supervised data acquisition and designed research. V.M. designed research, supervised data acquisition, performed and supervised data analysis and simulations, and wrote the manuscript.

Corresponding author

Correspondence to Victor Muñoz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Table 1 (PDF 521 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Campos, L., Liu, J., Wang, X. et al. A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy. Nat Methods 8, 143–146 (2011). https://doi.org/10.1038/nmeth.1553

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1553

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