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Title: Higher resolution in localization microscopy by slower switching of a photochromic protein
Authors: Mizuno, Hideaki ×
Dedecker, Peter
Ando, Ryoko
Fukano, Takashi
Hofkens, Johan
Miyawaki, Atsushi #
Issue Date: 2010
Publisher: Royal soc chemistry
Series Title: Photochemical & photobiological sciences vol:9 issue:2 pages:239-248
Abstract: Photoswitchable fluorophores play an essential role in super-resolution fluorescence microscopy, including techniques such as photoactivated localization microscopy (PALM). A determining factor in the precision of the images generated by PALM measurements is the photon numbers that can be detected from the fluorophores. Dronpa is a reversibly photoswitchable fluorescent protein that has been successfully used in PALM experiments. The number of photons per switching cycle that can be acquired for Dronpa depends on its off-switching rate, limiting the number of photons that can be recorded. In this study we report our discovery that the tetrameric ancestor of Dronpa, 22G, shows slower switching, and develop a mutant that displays switching kinetics between those of Dronpa and 22G. We show that the kinetics of the photoswitching are strongly related to self-association of the protein, supporting our view of dynamic flexibility as determining in the photoswitching. Similarly we find that higher-resolution PALM images can be acquired with slower-switching proteins due to their higher number of emitted photons per switching cycle.
ISSN: 1474-905X
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Molecular Imaging and Photonics
× corresponding author
# (joint) last author

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