Fluorescent proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state are important for innovative microscopy schemes, such as protein tracking1, fluorescence resonance energy transfer imaging2, sub-diffraction resolution microscopy3,4,5,6,7,8,9 and others. However, all available monomeric reversibly switchable fluorescent proteins (RSFPs) have similar properties and switching characteristics10,11,12, thereby limiting their use. Here, we introduce two bright green fluorescent RSFPs, bsDronpa and Padron, generated by extensive mutagenesis of the RSFP Dronpa10, with unique absorption and switching characteristics. Whereas bsDronpa features a broad absorption spectrum extending into the UV, Padron displays a switching behavior that is reversed to that of all green fluorescent RSFPs known to date. These two RSFPs enable live-cell fluorescence microscopy with multiple labels using a single detection color, because they can be distinguished by photoswitching. Furthermore, we demonstrate dual-color fluorescence microscopy with sub-diffraction resolution using bsDronpa and Dronpa whose emission maxima are separated by <20 nm.
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We thank S. Löbermann and J. Schilde for excellent technical assistance and J. Jethwa for carefully reading the manuscript. We acknowledge J.-W.L. de Gier, Stockholm University, Sweden, for providing us with the expression vector harboring the M13-GFP fusion.
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Andresen, M., Stiel, A., Fölling, J. et al. Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nat Biotechnol 26, 1035–1040 (2008). https://doi.org/10.1038/nbt.1493
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