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Cyanine fluorophore derivatives with enhanced photostability


Fluorescence applications requiring high photostability often depend on the use of solution additives to enhance fluorophore performance. Here we demonstrate that the direct or proximal conjugation of cyclooctatetraene (COT), 4-nitrobenzyl alcohol (NBA) or Trolox to the cyanine fluorophore Cy5 dramatically enhanced fluorophore photostability without otherwise affecting its native spectral characteristics. Such conjugation is a powerful means of improving the robustness of fluorescence-based applications demanding long-lived, nonblinking fluorescence emission.

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Figure 1: Enhancement of Cy5 photophysical properties through direct coupling to TSQs.
Figure 2: Role of proximity in the enhancement of Cy5 fluorescence with indirectly coupled TSQs.
Figure 3: Photostability of Cy5 and Cy5-TSQ conjugates in the presence of oxygen and in living cells.


  1. Aitken, C.E., Marshall, R.A. & Puglisi, J.D. Biophys. J. 94, 1826–1835 (2008).

    Article  CAS  Google Scholar 

  2. Dave, R., Terry, D.S., Munro, J.B. & Blanchard, S.C. Biophys. J. 96, 2371–2381 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Campos, L.A. et al. Nat. Methods 8, 143–146 (2011).

    Article  CAS  Google Scholar 

  5. Vogelsang, J. et al. Angew. Chem. Int. Edn Engl. 47, 5465–5469 (2008).

    Article  CAS  Google Scholar 

  6. Munro, J.B., Altman, R.B., O'Connor, N. & Blanchard, S.C. Mol. Cell 25, 505–517 (2007).

    Article  CAS  Google Scholar 

  7. Sakon, J.J. & Weninger, K.R. Nat. Methods 7, 203–205 (2010).

    Article  CAS  Google Scholar 

  8. Marling, J.B., Gregg, D.W. & Wood, L. Appl. Phys. Lett. 17, 527–530 (1970).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Albizu, L. et al. Nat. Chem. Biol. 6, 587–594 (2010).

    Article  CAS  Google Scholar 

  11. Blanchard, S.C. Curr. Opin. Struct. Biol. 19, 103–109 (2009).

    Article  CAS  Google Scholar 

  12. Diaspro, A., Chirico, G., Usai, C. & Ramoino, P. in Handbook of Biological Confocal Microscopy (ed., Pawley, J.B.) 690–702 (Springer, 2006).

  13. Cordes, T., Vogelsang, J. & Tinnefeld, P.O. J. Am. Chem. Soc. 131, 5018–5019 (2009).

    Article  CAS  Google Scholar 

  14. Vogelsang, J., Cordes, T., Forthmann, C., Steinhauer, C. & Tinnefeld, P. Proc. Natl. Acad. Sci. USA 106, 8107–8112 (2009).

    Article  CAS  Google Scholar 

  15. Qin, F. Biophys. J. 86, 1488–1501 (2004).

    Article  CAS  Google Scholar 

  16. Deerinck, T.J. J. Cell Biol. 126, 901–910 (1994).

    Article  CAS  Google Scholar 

  17. Hess, B. J. Chem. Theory Comput. 4, 435–447 (2008).

    Article  CAS  Google Scholar 

  18. Whitford, P.C. et al. Proteins 75, 430–441 (2008).

    Article  Google Scholar 

  19. Humphrey, W., Dalke, A. & Schulten, K. J. Mol. Graph. 14, 33–38 (1996).

    Article  CAS  Google Scholar 

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This work was supported by the Tri-Institutional Training Program in Chemical Biology, the Tri-Institutional Training Program in Computational Biology and Medicine, the Irma T. Hirschl/Monique Weill-Caulier Trust and the Lieber Center for Schizophrenia Research and Treatment at Columbia University. We thank J. Paige, M. Feldman, M. Wasserman and L. Wang for critical feedback on the manuscript, and P. Whitford for help with and discussions about performing molecular dynamics simulations.

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Authors and Affiliations



S.C.B., R.B.A. and D.S.T. designed in vitro experiments. Z.Z. and J.D.W. synthesized TSQ-conjugated fluorophores. R.B.A. made and purified complexes. R.B.A. performed single-molecule and bulk imaging experiments. Q.Z. performed bulk fluorescence and singlet-oxygen measurements. D.S.T. performed simulations. D.S.T., R.B.A. and S.C.B. analyzed in vitro data. S.C.B., J.A.J., and P.G. designed live-cell imaging experiments. Y.Z. and R.A.K. designed and constructed receptor constructs. P.G. performed the in vivo imaging and analyzed data. R.B.A., D.S.T. and P.G. designed figures. R.B.A. and S.C.B. wrote the manuscript, which all authors edited.

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Correspondence to Scott C Blanchard.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Note 1 (PDF 576 kb)

Supplementary Video 1

Photostability of Cy5-TSQ conjugates when linked to DNA duplexes. Shown are single-molecule TIRF movies of Cy5 (top left), Cy5-COT (top right), Cy5-NBA (bottom left), Cy5-Trolox (bottom right) taken at 10 frames s−1 with oxygen scavenging (as in Fig. 1). The movie is played at five times actual imaging speed. The elapsed time (in min:s) is displayed in the top left corner. (AVI 26138 kb)

Supplementary Video 2

Photostability of Cy5 and Cy5-COT–labeled dopamine D2 receptors (D2Rs) in living CHO cells (top left). Single-molecule TIRF video sequence of Cy5 labeled D2Rs. In a deoxygenated environment Cy5-labeled D2 receptors show enhanced photostability at the cost of higher blinking rates (top right). A direct linkage of COT to Cy5 improves the photostability (bottom left). The greatest photostability of D2Rs was observed by labeling with Cy5-COT and imaging in a deoxygenated environment (bottom right). All image sequences were recorded at a rate of 25 frames s−1. (AVI 5413 kb)

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Altman, R., Terry, D., Zhou, Z. et al. Cyanine fluorophore derivatives with enhanced photostability. Nat Methods 9, 68–71 (2012).

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