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Improving the photostability of bright monomeric orange and red fluorescent proteins

Nature Methods volume 5, pages 545–551 (2008)Cite this article

Abstract

All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

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Figure 1: Comparison of photobleaching curves.
Figure 2: Fluorescence imaging of TagRFP-T subcellular targeting fusions.
Figure 3: Widefield fluorescence imaging of mOrange2 subcellular targeting fusions.

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Acknowledgements

L.A. Gross performed mass spectroscopy. S.R. Adams performed gel filtration experiments. We thank R.E. Campbell and C.T. Dooley for helpful discussion. Sequencing services were provided by the University of California, San Diego Cancer Center shared sequencing resource and the Florida State University Bioanalytical and Molecular Cloning DNA Sequencing Laboratory. N.C.S. was a Howard Hughes Medical Institute predoctoral fellow during this work. This work was additionally supported by the US National Institutes of Health (NS27177 and GM72033) and the Howard Hughes Medical Institute.

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  1. Nathan C Shaner

    Present address: Present address: The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, California 92037, USA.,

Authors and Affiliations

  1. Department of Pharmacology, University of California at San Diego, 9500 Gilman Drive, La Jolla, 92093, California, USA

    Nathan C Shaner, Michael Z Lin, Michael R McKeown, Paul A Steinbach & Roger Y Tsien

  2. Howard Hughes Medical Institute, University of California at San Diego, 9500 Gilman Drive, La Jolla, 92093, California, USA

    Michael Z Lin, Michael R McKeown, Paul A Steinbach & Roger Y Tsien

  3. Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, 92093, California, USA

    Roger Y Tsien

  4. National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, 1800 East Paul Dirac Drive, Tallahassee, 32310, Florida, USA

    Kristin L Hazelwood & Michael W Davidson

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Contributions

N.C.S. designed the photostability selection protocol, performed all directed evolution and physical characterization of mApple and mOrange2, analyzed and organized all data collected by other authors, and prepared the manuscript; M.Z.L. and M.R.M. performed directed evolution and physical characterization of TagRFP-T; P.A.S. designed the home-built components of the solar simulator apparatus and performed photobleaching measurements of purified proteins; K.L.H. and M.W.D. constructed mammalian expression vectors and performed all microscopy experiments involving live cells; R.Y.T. contributed to conceptual development, data analysis and manuscript preparation; all authors contributed to editing the manuscript.

Corresponding author

Correspondence to Roger Y Tsien.

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Competing interests

N.C.S., M.Z.L., M.R.M. and R.Y.T. are listed as inventors in a US patent application assigned to the University of California, which includes as claims the fluorescent proteins described in this manuscript.

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Supplementary Figure 1, Supplementary Notes 1–3, Supplementary Methods (PDF 2180 kb)

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Shaner, N., Lin, M., McKeown, M. et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5, 545–551 (2008). https://doi.org/10.1038/nmeth.1209

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