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Fast and Reversible Photoswitching of the Fluorescent Protein Dronpa as Evidenced by Fluorescence Correlation Spectroscopy

Peter Dedecker ^*, Jun-ichi Hotta ^*, Ryoko Ando , Atsushi Miyawaki , Yves Engelborghs  and Johan Hofkens ^*

^* Laboratory of Photochemistry and Spectroscopy, Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium; Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2–1 Hirosawa, Wako-City, Saitama, 351-0198, Japan; and Laboratory for Biomolecular Dynamics, Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium

Correspondence: Address reprint requests and inquiries to Johan Hofkens, E-mail: johan.hofkens{at}chem.kuleuven.be.

Controlling molecular properties through photoirradiation holds great promise for its potential for noninvasive and selective manipulation of matter. Photochromism has been observed for several different molecules, including green fluorescent proteins, and recently the discovery of a novel photoswitchable green fluorescent protein called Dronpa was reported. Dronpa displays reversible and highly efficient on/off photoswitching of its fluorescence emission, and reversible switching of immobilized single molecules of Dronpa with response times faster than 20 ms was demonstrated. In this Letter, we expand these observations to freely diffusing molecules by using fluorescence correlation spectroscopy with simultaneous excitation at 488 and 405 nm. By varying the intensity of irradiation at 405 nm, we demonstrate the reversible photoswitching of Dronpa under these conditions, and from the obtained autocorrelation functions we conclude that this photoswitching can occur within tens of microseconds.

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