A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants.
The photophysical properties and photoswitching scheme of the reversible photoswitchable green fluorescent protein-like fluorescent proteins Dronpa-2 and Dronpa-3 were investigated by means of ensemble and single-molecule fluorescence spectroscopy and compared to those of the precursor protein Dronpa. The faster response to light and the faster dark recovery of the new mutants observed in bulk also hold at the single-molecule level. Analysis of the single-molecule traces allows us to extract the efficiencies and rate constants of the pathways involved in the forward and backward switching, and we find important differences when comparing the mutants to Dronpa. We rationalize our results in terms of a higher conformational freedom of the chromophore in the protein environment provided by the beta-can. This thorough understanding of the photophysical parameters has allowed us to optimize the acquisition parameters for camera-based sub-diffraction-limit imaging with these photochromic proteins. We show that Dronpa and its mutants are useful for fast photoactivation-localization microscopy (PALM) using common wide-field microscopy equipment, as individual fluorescent proteins can be localized several times. We provide a new approach to achieve fast PALM by introducing simultaneous two-color stroboscopic illumination.
Flors
Cristina
C
Department of Chemistry and Institute for Nanoscale Physics and Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium.
Hotta
Jun-ichi
J
Uji-i
Hiroshi
H
Dedecker
Peter
P
Ando
Ryoko
R
Mizuno
Hideaki
H
Miyawaki
Atsushi
A
Hofkens
Johan
J
D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D018613:Microscopy, Confocal / Q000379:methods; D008958:Models, Molecular; D009154:Mutation; D010777:Photochemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors
D049452:Green Fluorescent Proteins