Title: A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants
Authors: Flors Ong, Cristina ×
Hotta, Jun-ichi
Uji-I, Hiroshi
Dedecker, Peter
Ando, Ryoko
Mizuno, Hideaki
Miyawaki, Atsushi
Hofkens, Johan #
Issue Date: Nov-2007
Publisher: American Chemical Society
Series Title: Journal of the American Chemical Society vol:129 issue:45 pages:13970-13977
Abstract: 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 P-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.
ISSN: 0002-7863
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Molecular Imaging and Photonics
Biochemistry, Molecular and Structural Biology Section
× corresponding author
# (joint) last author

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