Physics

Cell

Enzyme

Biology

Optics

Biophysics

Live cell imaging

Catalysis

Nucleophile

Chemistry

Fluorophore

Xanthene

Rhodamine

Glutathione

Biochemistry

Photochemistry

Intracellular

Fluorescence

Journal of the American Chemical Society

Single-molecule localization microscopy (SMLM) allows the reconstruction of super-resolution images but generally requires prior intense laser irradiation and in some cases additives to induce blinking of conventional fluorophores. We previously introduced a spontaneously blinking rhodamine fluorophore based on an intramolecular spirocyclization reaction for live-cell SMLM under physiological conditions. Here, we report a novel principle of spontaneous blinking in living cells, which utilizes reversible ground-state nucleophilic attack of intracellular glutathione (GSH) upon a xanthene fluorophore. Structural optimization afforded two pyronine fluorophores with different colors, both of which exhibit equilibrium (between the fluorescent dissociated form and the nonfluorescent GSH adduct form) and blinking kinetics that enable SMLM of microtubules or mitochondria in living cells. Furthermore, by using spontaneously blinking fluorophores working in the near-infrared (NIR) and green ranges, we succeeded in dual-color live-cell SMLM without the need for optimization of the imaging medium.

Spontaneously Blinking Fluorophores Based on Nucleophilic Addition/Dissociation of Intracellular Glutathione for Live-Cell Super-resolution Imaging