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.

Photoactivatable mCherry for high-resolution two-color fluorescence microscopy

An Erratum to this article was published on 01 April 2009

This article has been updated


The reliance of modern microscopy techniques on photoactivatable fluorescent proteins prompted development of mCherry variants that are initially dark but become red fluorescent after violet-light irradiation. Using ensemble and single-molecule characteristics as selection criteria, we developed PAmCherry1 with excitation/emission maxima at 564/595 nm. Compared to other monomeric red photoactivatable proteins, it has faster maturation, better pH stability, faster photoactivation, higher photoactivation contrast and better photostability. Lack of green fluorescence and single-molecule behavior make monomeric PAmCherry1 a preferred tag for two-color diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two-color photoactivated localization microscopy (PALM). We performed PALM imaging using PAmCherry1-tagged transferrin receptor expressed alone or with photoactivatable GFP–tagged clathrin light chain. Pair correlation and cluster analyses of the resulting PALM images identified ≤200 nm clusters of transferrin receptor and clathrin light chain at ≤25 nm resolution and confirmed the utility of PAmCherry1 as an intracellular probe.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: Spectral, biochemical and photochemical properties of the purified PAmCherry variants.
Figure 2: Plasma membrane distribution of TfR observed using PALM.
Figure 3: Comparison of PAmCherry1 and tdEosFP fusions in fixed cells.
Figure 4: Distributions of TfR and CLC by two-color PALM.

Change history

  • 26 February 2009

    NOTE: In the version of this article initially published, the affiliations listed for Fedor V. Subach were incorrect. The error has been corrected in the HTML and PDF versions of the article.


  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).

    Article  CAS  Google Scholar 

  2. Hell, S.W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Hess, S.T., Girirajan, T.P. & Mason, M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    Article  CAS  Google Scholar 

  5. Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  6. Egner, A. et al. Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys. J. 93, 3285–3290 (2007).

    Article  CAS  Google Scholar 

  7. Flors, C. et al. A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants. J. Am. Chem. Soc. 129, 13970–13977 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. 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).

    Article  CAS  Google Scholar 

  10. Chudakov, D.M. et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21, 191–194 (2003).

    Article  CAS  Google Scholar 

  11. Wiedenmann, J. et al. EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc. Natl. Acad. Sci. USA 101, 15905–15910 (2004).

    Article  CAS  Google Scholar 

  12. Gurskaya, N.G. et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat. Biotechnol. 24, 461–465 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Ando, R., Mizuno, H. & Miyawaki, A. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306, 1370–1373 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. 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).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. Verkhusha, V.V. & Lukyanov, K.A. The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat. Biotechnol. 22, 289–296 (2004).

    Article  CAS  Google Scholar 

  20. Huebers, H.A., Huebers, E., Josephson, B. & Csiba, E. A highly efficient chemical isolation procedure for the rat placental transferrin receptor. Biochim. Biophys. Acta 991, 30–35 (1989).

    Article  CAS  Google Scholar 

  21. Gallione, C.J. & Rose, J.K. A single amino acid substitution in a hydrophobic domain causes temperature sensitive cell-surface transport of a mutant viral glycoprotein. J. Virol. 54, 374–382 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. McGraw, T.E. & Maxfield, F.R. Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain. Cell Regul. 1, 369–377 (1990).

    Article  CAS  Google Scholar 

  23. Hopkins, C.R. The appearance and internalization of transferrin receptors at the margins of spreading human tumor cells. Cell 40, 199–208 (1985).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Pearse, B.M.F. & Robinson, M.S. Clathrin, adaptors, and sorting. Annu. Rev. Cell Biol. 6, 151–171 (1990).

    Article  CAS  Google Scholar 

  27. Heuser, J.E. & Anderson, R.G. Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J. Cell Biol. 108, 389–400 (1989).

    Article  CAS  Google Scholar 

  28. Kirchhausen, T. Clathrin. Annu. Rev. Biochem. 69, 699–727 (2000).

    Article  CAS  Google Scholar 

  29. Habuchi, S. et al. Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc. Natl. Acad. Sci. USA 102, 9511–9516 (2005).

    Article  CAS  Google Scholar 

  30. Bock, H. et al. Two-color far-field fluorescence nanoscopy based on photoswitchable emitters. Appl. Phys. B 88, 161–165 (2007).

    Article  CAS  Google Scholar 

Download references


We thank R. Tsien (University of California at San Diego) for providing the pRSETB-mCherry plasmid, J. Wiedenmann (University of Ulm) for providing plasmids encoding EosFP variants, J. Zhang for assistance with flow cytometry, O. Subach for assistance with cell culture and imaging, and E. Betzig and H. Hess for assistance with PALM experiments, analysis and discussion. This work was supported by grants GM070358 and GM073913 from the US National Institutes of Health to V.V.V.

Author information

Authors and Affiliations



F.V.S. developed proteins and characterized them in vitro. G.H.P., S.M. and J.M.G. characterized proteins in mammalian cells. J.L.-S. and V.V.V. designed and planned the project. G.H.P. and V.V.V. wrote the manuscript.

Corresponding author

Correspondence to Vladislav V Verkhusha.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1, Supplementary Methods (PDF 9095 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Subach, F., Patterson, G., Manley, S. et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Methods 6, 153–159 (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