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Photoactivatable mCherry for high-resolution two-color fluorescence microscopy

Nature Methods volume 6, pages 153–159 (2009)Cite this article

An Erratum to this article was published on 01 April 2009

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Abstract

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.

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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.

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  • 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.

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Acknowledgements

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.

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Author notes

  1. Fedor V Subach and George H Patterson: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, 10461, New York, USA

    Fedor V Subach & Vladislav V Verkhusha

  2. Section on Organelle Biology, Cell Biology and Metabolism Program, National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bethesda, 20892, Maryland, USA

    George H Patterson, Suliana Manley, Jennifer M Gillette & Jennifer Lippincott-Schwartz

Authors
  1. Fedor V Subach
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  2. George H Patterson
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  3. Suliana Manley
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  4. Jennifer M Gillette
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  5. Jennifer Lippincott-Schwartz
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  6. Vladislav V Verkhusha
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Contributions

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.

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

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Subach, F., Patterson, G., Manley, S. et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Methods 6, 153–159 (2009). https://doi.org/10.1038/nmeth.1298

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