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Issue 2, 2010
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Higher resolution in localizationmicroscopy by slower switching of a photochromic protein

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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.

Graphical abstract: Higher resolution in localization microscopy by slower switching of a photochromic protein

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Article information


Submitted
06 Oct 2009
Accepted
11 Nov 2009
First published
18 Jan 2010

Photochem. Photobiol. Sci., 2010,9, 239-248
Article type
Paper

Higher resolution in localization microscopy by slower switching of a photochromic protein

H. Mizuno, P. Dedecker, R. Ando, T. Fukano, J. Hofkens and A. Miyawaki, Photochem. Photobiol. Sci., 2010, 9, 239
DOI: 10.1039/B9PP00124G

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