Persistent Fluorescence Switching of a Probe Using a Photochromic Quencher with High Photostability Assisted by Protein-Surface Modification.

Photoswitchable fluorescent molecules (PSFMs) are widely applicable in the life sciences for super-resolution imaging. Owing to the large and hydrophobic molecular structures of PSFMs that may aggregate in a biological medium, the development of synthetic PSFMs with persistent reversible photoswitching is challenging. Here, we established a protein-surface-assisted photoswitching strategy that allows for persistent reversible fluorescence photoswitching of a PSFM in an aqueous solution. As a first step, we applied the photochromic chromophore furylfulgimide (FF) as a photoswitchable fluorescence quencher and developed a Förster resonance energy transfer-based PSFM, named FF-TMR. Most importantly, the protein-surface modification strategy allows FF-TMR to exhibit persistent reversible photoswitching performance in an aqueous solution. In fixed cells, the fluorescence intensity of FF-TMR bound to antitubulin antibody was repetitively modulated. The protein-surface-assisted photoswitching strategy will be a useful platform to broaden the utility of functionalized synthetic chromophores enabling persistent fluorescence switching that inherits their high resistance to light irradiation.