Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy.
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.
Andresen
Martin
M
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Stiel
Andre C
AC
Fölling
Jonas
J
Wenzel
Dirk
D
Schönle
Andreas
A
Egner
Alexander
A
Eggeling
Christian
C
Hell
Stefan W
SW
Jakobs
Stefan
S
D001709:Biotechnology / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D021621:Imaging, Three-Dimensional; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D053758:Nanoparticles / Q000737:chemistry; D036103:Nanotechnology / Q000379:methods*; D010777:Photochemistry / Q000379:methods; D010957:Plasmids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014466:Ultraviolet Rays
D008164:Luminescent Proteins