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.
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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.).
The authors declare no competing financial interests.
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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