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Nonblinking and long-lasting single-molecule fluorescence imaging

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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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Figure 1: Effects of BME and Trolox on the photophysics of single DNA-conjugated Cy5 molecules.
Figure 2: Comparison of BME and Trolox for single-molecule FRET experiments.

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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 to Ivan Rasnik.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Single molecule FRET experiments in the presence of 2 mM Trolox or 143 mM BME for the construct of Figure 2d. (PDF 38 kb)

Supplementary Methods (PDF 58 kb)

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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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