A photoswitchable orange-to-far-red fluorescent protein, PSmOrange


We report a photoswitchable monomeric Orange (PSmOrange) protein that is initially orange (excitation, 548 nm; emission, 565 nm) but becomes far-red (excitation, 636 nm; emission, 662 nm) after irradiation with blue-green light. Compared to its parental orange proteins, PSmOrange has greater brightness, faster maturation, higher photoconversion contrast and better photostability. The red-shifted spectra of both forms of PSmOrange enable its simultaneous use with cyan-to-green photoswitchable proteins to study four intracellular populations. Photoconverted PSmOrange has, to our knowledge, the most far-red excitation peak of all GFP-like fluorescent proteins, provides diffraction-limited and super-resolution imaging in the far-red light range, is optimally excited with common red lasers, and can be photoconverted subcutaneously in a mouse. PSmOrange photoswitching occurs via a two-step photo-oxidation process, which causes cleavage of the polypeptide backbone. The far-red fluorescence of photoconverted PSmOrange results from a new chromophore containing N-acylimine with a co-planar carbon-oxygen double bond.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Characterization of purified PSmOrange in vitro.
Figure 2: Imaging of PSmOrange in mammalian cells.
Figure 3: Imaging of PSmOrange in vivo.
Figure 4: Mass spectrometry analysis of the PSmOrange chromophore.


  1. 1

    Lukyanov, K.A., Chudakov, D.M., Lukyanov, S. & Verkhusha, V.V. Innovation: photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. 6, 885–891 (2005).

    CAS  Article  Google Scholar 

  2. 2

    Patterson, G.H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002).

    CAS  Article  Google Scholar 

  3. 3

    Subach, F.V. et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat. Methods 6, 153–159 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Subach, F.V. et al. Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. J. Am. Chem. Soc. 132, 6481–6491 (2010).

    CAS  Article  Google Scholar 

  5. 5

    Chudakov, D.M., Lukyanov, S. & Lukyanov, K.A. Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2. Nat. Protoc. 2, 2024–2032 (2007).

    CAS  Article  Google Scholar 

  6. 6

    McKinney, S.A. et al. A bright and photostable photoconvertible fluorescent protein. Nat. Methods 6, 131–133 (2009).

    CAS  Article  Google Scholar 

  7. 7

    Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. & Miyawaki, A. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 12651–12656 (2002).

    CAS  Article  Google Scholar 

  8. 8

    Habuchi, S., Tsutsui, H., Kochaniak, A.B., Miyawaki, A. & van Oijen, A.M. mKikGR, a monomeric photoswitchable fluorescent protein. PLoS ONE 3, e3944 (2008).

    Article  Google Scholar 

  9. 9

    Hoi, H. et al. A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization. J. Mol. Biol. 401, 776–791 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Toomre, D. & Bewersdorf, J. A new wave of cellular imaging. Annu. Rev. Cell Dev. Biol. 26, 285–314 (2010).

    CAS  Article  Google Scholar 

  11. 11

    Stiel, A.C. et al. Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy. Biophys. J. 95, 2989–2997 (2008).

    CAS  Article  Google Scholar 

  12. 12

    Folling, J. et al. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat. Methods 5, 943–945 (2008).

    Article  Google Scholar 

  13. 13

    Lim, Y.T. et al. Selection of quantum dot wavelengths for biomedical assays and imaging. Mol. Imaging 2, 50–64 (2003).

    CAS  Article  Google Scholar 

  14. 14

    Deliolanis, N.C. et al. Performance of the red-shifted fluorescent proteins in deep tissue molecular imaging applications. J. Biomed. Opt. 13, 044008 (2008).

    Article  Google Scholar 

  15. 15

    Morozova, K.S. et al. Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscory. Biophys. J. 99, L13–L15 (2010).

    CAS  Article  Google Scholar 

  16. 16

    Strack, R.L. et al. A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling. Biochemistry 48, 8279–8281 (2009).

    CAS  Article  Google Scholar 

  17. 17

    Shcherbo, D. et al. Far-red fluorescent tags for protein imaging in living tissues. Biochem. J. 418, 567–574 (2009).

    CAS  Article  Google Scholar 

  18. 18

    Lin, M.Z. et al. Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem. Biol. 16, 1169–1179 (2009).

    CAS  Article  Google Scholar 

  19. 19

    Shcherbo, D. et al. Near-infrared fluorescent proteins. Nat. Methods 7, 827–829 (2010).

    CAS  Article  Google Scholar 

  20. 20

    Kremers, G.J., Hazelwood, K.L., Murphy, C.S., Davidson, M.W. & Piston, D.W. Photoconversion in orange and red fluorescent proteins. Nat. Methods 6, 355–358 (2009).

    CAS  Article  Google Scholar 

  21. 21

    Bogdanov, A.M. et al. Green fluorescent proteins are light-induced electron donors. Nat. Chem. Biol. 5, 459–461 (2009).

    CAS  Article  Google Scholar 

  22. 22

    Shaner, N.C. et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods 5, 545–551 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Strack, R.L. et al. A non-cytotoxic DsRed variant for whole-cell labeling. Nat. Methods 5, 955–957 (2008).

    CAS  Article  Google Scholar 

  24. 24

    Etienne-Manneville, S. From signaling pathways to microtubule dynamics: the key players. Curr. Opin. Cell Biol. 22, 104–111 (2010).

    CAS  Article  Google Scholar 

  25. 25

    Kuo, C., Coquoz, O., Troy, T.L., Xu, H. & Rice, B.W. Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging. J. Biomed. Opt. 12, 024007 (2007).

    Article  Google Scholar 

  26. 26

    Smith, A.M., Mancini, M.C. & Nie, S. Bioimaging: second window for in vivo imaging. Nat. Nanotechnol. 4, 710–711 (2009).

    CAS  Article  Google Scholar 

  27. 27

    Shu, X., Shaner, N.C., Yarbrough, C.A., Tsien, R.Y. & Remington, S.J. Novel chromophores and buried charges control color in mFruits. Biochemistry 45, 9639–9647 (2006).

    CAS  Article  Google Scholar 

  28. 28

    Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).

    Article  Google Scholar 

  29. 29

    Yampolsky, I.V. et al. Synthesis and properties of the chromophore of the asFP595 chromoprotein from Anemonia sulcata. Biochemistry 44, 5788–5793 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Tojo, G. & Fernández, M. Oxidation of Alcohols to Aldehydes and Ketones (Springer, New York, 2006).

  31. 31

    Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    CAS  Article  Google Scholar 

  32. 32

    Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    CAS  Article  Google Scholar 

  33. 33

    Chudakov, D.M. et al. Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22, 1435–1439 (2004).

    CAS  Article  Google Scholar 

  34. 34

    Verkhusha, V.V. & Sorkin, A. Conversion of the monomeric red fluorescent protein into a photoactivatable probe. Chem. Biol. 12, 279–285 (2005).

    CAS  Article  Google Scholar 

  35. 35

    Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    CAS  Article  Google Scholar 

  36. 36

    Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008).

    CAS  Article  Google Scholar 

  37. 37

    Shroff, H. et al. Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc. Natl. Acad. Sci. USA 104, 20308–20313 (2007).

    CAS  Article  Google Scholar 

Download references


We thank J. Zhang and L. Tesfa for assistance with flow cytometry, H. Xiao for help with mass-spectrometry analysis, K. Kim and G. Filonov for assistance with mouse experiments and useful discussions, M. Davidson (Florida State University) for vimentin, keratin, myosin and paxillin plasmids, and B. Glick (University of Chicago), D. Chudakov and K. Lukyanov (both from Institute of Bioorganic Chemistry) for plasmids encoding far-red fluorescent proteins. This work was supported by US National Institutes of Health (GM073913 to V.V.V. and CA100324 to J.C.) and by the National Institutes of Health Intramural Research Program including the National Institute of Biomedical Imaging and Bioengineering (to G.H.P.).

Author information




O.M.S. developed the protein and characterized it in vitro. O.M.S. and G.H.P. characterized the protein in mammalian cells. O.M.S. and L.-M.T. characterized the protein in mouse models. O.M.S., Y.W. and J.S.C. performed tumor experiments. V.V.V. designed the project and, together with O.M.S., planned and discussed the project, and wrote the manuscript.

Corresponding author

Correspondence to Vladislav V Verkhusha.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Tables 1–2 (PDF 3947 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Subach, O., Patterson, G., Ting, L. et al. A photoswitchable orange-to-far-red fluorescent protein, PSmOrange. Nat Methods 8, 771–777 (2011). https://doi.org/10.1038/nmeth.1664

Download citation


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