Skip to main content

Thank you for visiting 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.

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

Download references


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

Authors and Affiliations



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.

Corresponding author

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kremers, GJ., Hazelwood, K., Murphy, C. et al. Photoconversion in orange and red fluorescent proteins. Nat Methods 6, 355–358 (2009).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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