Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy.
Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at approximately 610 nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the on-to-off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules.
Stiel
Andre C
AC
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Andresen
Martin
M
Bock
Hannes
H
Hilbert
Michael
M
Schilde
Jessica
J
Schönle
Andreas
A
Eggeling
Christian
C
Egner
Alexander
A
Hell
Stefan W
SW
Jakobs
Stefan
S
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002417:Cattle; D003116:Color; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D008027:Light; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D008853:Microscopy; D036103:Nanotechnology; D015202:Protein Engineering / Q000379:methods*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D000097573:Red Fluorescent Protein
D015415:Biomarkers; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein