Nonblinking and long-lasting single-molecule fluorescence imaging (original) (raw)

Nature Methods volume 3, pages 891–893 (2006)Cite this article

Abstract

Photobleaching and blinking of fluorophores pose fundamental limitations on the information content of single-molecule fluorescence measurements. Photoinduced blinking of Cy5 has hampered many previous investigations using this popular fluorophore. Here we show that Trolox in combination with the enzymatic oxygen-scavenging system eliminates Cy5 blinking, dramatically reduces photobleaching and improves the signal linearity at high excitation rates, significantly extending the applicability of single-molecule fluorescence techniques.

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References

  1. Weiss, S. Science 283, 1676–1683 (1999).
    Article CAS Google Scholar
  2. Ha, T. Methods 25, 78–86 (2001).
    Article CAS Google Scholar
  3. Widengren, J. & Schwille, P. J. Phys. Chem. A 104, 6416–6428 (2000).
    Article CAS Google Scholar
  4. Fureder-Kitzmuller, E., Hesse, J., Ebner, A., Gruber, H.J. & Schutz, G.J. Chem. Phys. Lett. 404, 13–18 (2005).
    Article Google Scholar
  5. Sabanayagam, C.R., Eid, J.S. & Meller, A. J. Chem. Phys. 123, 224708 (2005).
    Article Google Scholar
  6. Benesch, R.E. & Benesch, R. Science 118, 447–448 (1953).
    Article CAS Google Scholar
  7. Kishino, A. & Yanagida, T. Nature 334, 74–76 (1988).
    Article CAS Google Scholar
  8. Bates, M., Blosser, T.R. & Zhuang, X.W. Phys. Rev. Lett. 94, 108101 (2005).
    Article Google Scholar
  9. Ha, T.J. et al. Chem. Phys. 247, 107–118 (1999).
    Article CAS Google Scholar
  10. Heilemann, M., Margeat, E., Kasper, R., Sauer, M. & Tinnefeld, P. J. Am. Chem. Soc. 127, 3801–3806 (2005).
    Article CAS Google Scholar
  11. Blanchard, S.C., Kim, H.D., Gonzalez, R.L., Puglisi, J.D. & Chu, S. Proc. Natl. Acad. Sci. USA 101, 12893–12898 (2004).
    Article CAS Google Scholar
  12. Grunwell, J.R. et al. J. Am. Chem. Soc. 123, 4295–4303 (2001).
    Article CAS Google Scholar
  13. McKinney, S.A., Declais, A.C., Lilley, D.M. & Ha, T. Nat. Struct. Biol. 10, 93–97 (2003).
    Article CAS Google Scholar
  14. Vrljic, M., Nishimura, S.Y., Brasselet, S., Moerner, W.E. & McConnell, H.M. Biophys. J. 83, 2681–2692 (2002).
    Article CAS Google Scholar
  15. Churchman, L.S., Okten, Z., Rock, R.S., Dawson, J.F. & Spudich, J.A. Proc. Natl. Acad. Sci. USA 102, 1419–1423 (2005).
    Article CAS Google Scholar

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Acknowledgements

We thank J. Liu for introducing Trolox to us and the US National Institutes of Health for grants.

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Authors and Affiliations

  1. Department of Physics, Emory University, Atlanta, 30322, Georgia, USA
    Ivan Rasnik
  2. Department of Physics, University of Illinois, Urbana, 61801, Illinois, USA
    Sean A McKinney & Taekjip Ha
  3. Howard Hughes Medical Institute, Urbana, 61801, Illinois, USA
    Taekjip Ha

Authors

  1. Ivan Rasnik
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  2. Sean A McKinney
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  3. Taekjip Ha
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Correspondence toIvan Rasnik.

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Rasnik, I., McKinney, S. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging.Nat Methods 3, 891–893 (2006). https://doi.org/10.1038/nmeth934

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