Reactivatable stimulated emission depletion microscopy using fluorescence-recoverable nanographene

Stimulated emission depletion (STED) microscopy, a key optical super-resolution imaging method, has extended our ability to view details to resolution levels of tens of nanometers. Its resolution depends on fluorophore de-excitation efficiency, and increases with depletion laser power. However, high-power irradiation permanently turns off the fluorescence due to photo-bleaching of the fluorophores. As a result, there is a trade-off between spatial resolution and imaging time. Here, we overcome this limitation by introducing reactivatable STED (ReSTED) based on the photophysical properties of the nanographene dibenzo[hi,st]ovalene (DBOV). In contrast to the photo-induced decomposition of other fluorophores, the fluorescence of DBOV is only temporarily deactivated and can be reactivated by near-infrared light (including the 775 nm depletion beam). As a result, this fluorophore allows for hours-long, high-resolution 3D STED imaging, greatly expanding the applications of STED microscopy.