Photoconversion in orange and red fluorescent proteins


We found that photoconversion is fairly common among orange and red fluorescent proteins, as in a screen of 12 proteins, 8 exhibited photoconversion. Specifically, three red fluorescent proteins could be switched to a green state, and two orange variants could be photoconverted to a far-red state. The orange proteins are ideal for dual-probe highlighter applications, and they exhibited the most red-shifted excitation of all fluorescent proteins described to date.

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Figure 1: Photoconversion of mKate, HcRed1 and mOrange1 in HeLa cells.
Figure 2: mOrange1 and Kaede photoconversion rates.
Figure 3: Dual-probe optical highlighting with mOrange1 and Dronpa.


  1. 1

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

  2. 2

    Chudakov, D.M. et al. Nat. Biotechnol. 22, 1435–1439 (2004).

  3. 3

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

  4. 4

    Chudakov, D.M., Lukyanov, S. & Lukyanov, K.A. Nat. Protocols 2, 2024–2032 (2007).

  5. 5

    Wiedenmann, J. et al. Proc. Natl. Acad. Sci. USA 101, 15905–15910 (2004).

  6. 6

    Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. & Miyawaki, A. Proc. Natl. Acad. Sci. USA 99, 12651–12656 (2002).

  7. 7

    Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N. & Miyawaki, A. EMBO Rep. 6, 233–238 (2005).

  8. 8

    Shcherbo, D. et al. Nat. Methods 4, 741–746 (2007).

  9. 9

    Merzlyak, E.M. et al. Nat. Methods 4, 555–557 (2007).

  10. 10

    Shaner, N.C. et al. Nat. Methods 5, 545–551 (2008).

  11. 11

    Gurskaya, N.G. et al. FEBS Lett. 507, 16–20 (2001).

  12. 12

    Shaner, N.C. et al. Nat. Biotechnol. 22, 1567–1572 (2004).

  13. 13

    Karasawa, S., Araki, T., Nagai, T., Mizuno, H. & Miyawaki, A. Biochem. J. 381, 307–312 (2004).

  14. 14

    Goedhart, J., Vermeer, J.E., Adjobo-Hermans, M.J., van Weeren, L. & Gadella, T.W. Jr. PLoS ONE 2, e1011 (2007).

  15. 15

    Dittrich, P.S., Schafer, S.P. & Schwille, P. Biophys. J. 89, 3446–3455 (2005).

  16. 16

    Habuchi, S. et al. Proc. Natl. Acad. Sci. USA 102, 9511–9516 (2005).

  17. 17

    Marchant, J.S., Stutzmann, G.E., Leissring, M.A., LaFerla, F.M. & Parker, I. Nat. Biotechnol. 19, 645–649 (2001).

  18. 18

    Habuchi, S. et al. J. Am. Chem. Soc. 127, 8977–8984 (2005).

  19. 19

    Valentin, G. et al. Nat. Methods 2, 801 (2005).

  20. 20

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

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We thank D.M. Chudakov (Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry,), A. Miyawaki (RIKEN Brain Science Institute) and R.Y. Tsien (University of California at San Diego) for providing plasmids encoding fluorescent protein variants. Measurements were performed in part at the Vanderbilt University Medical Center Cell Imaging Shared Resource (National Institutes of Health grants CA68485, DK20593, DK58404) and at the Quantitative Fluorescence Microcopy course at Mount Desert Island Biological Lab. We thank G. Daniels (Leica), J. Wailes (Zeiss), B. Burklow (Olympus) and S. Schwartz (Nikon) for assistance with their microscopes, and B. Livesay for help with the mVenus experiments. This work was supported by National Institutes of Health grant GM72048 (to D.W.P.).

Author information

G.J.K. designed the experiments, performed part of the microscopy experiments, analyzed and organized all data collected by other authors, and prepared the manuscript; K.L.H. and C.S.M. constructed mammalian expression vectors and performed part of the microscopy experiments; M.W.D. constructed mammalian expression vectors and contributed to editing the manuscript; D.W.P. contributed to the conceptual design and manuscript preparation.

Correspondence to David W Piston.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-4 and Supplementary Notes 1-2 (PDF 1688 kb)

Supplementary Video 1

Optical highlighting with mOrange2 during mitosis. Time-lapse imaging of a HeLa cell expressing H2B-mOrange2 going through mitosis after part of the nucleus was photoconverted during prophase. (MOV 1221 kb)

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Kremers, G., Hazelwood, K., Murphy, C. et al. Photoconversion in orange and red fluorescent proteins. Nat Methods 6, 355–358 (2009) doi:10.1038/nmeth.1319

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