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Fluorescent proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state are important for innovative microscopy schemes, such as protein tracking, fluorescence resonance energy transfer imaging, sub-diffraction resolution microscopy and others. However, all available monomeric reversibly switchable fluorescent proteins (RSFPs) have similar properties and switching characteristics, thereby limiting their use. Here, we introduce two bright green fluorescent RSFPs, bsDronpa and Padron, generated by extensive mutagenesis of the RSFP Dronpa, 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.

Original publication

DOI

10.1038/nbt.1493

Type

Journal article

Journal

Nat Biotechnol

Publication Date

09/2008

Volume

26

Pages

1035 - 1040

Keywords

Biotechnology, Escherichia coli, Fluorescence, Imaging, Three-Dimensional, Light, Luminescent Proteins, Microscopy, Confocal, Microscopy, Fluorescence, Mutagenesis, Site-Directed, Nanoparticles, Nanotechnology, Photochemistry, Plasmids, Saccharomyces cerevisiae, Ultraviolet Rays