Single-visible-light performed STORM imaging with activatable photoswitches

Stochastic optical reconstruction microscopy (STORM) overcomes the diffraction limit of optical imaging, facilitating high-resolution visualization of cellular substructures at the nanoscale. Essential to this technique is the development of fluorescent photoswitches. However, existing photoswitches typically rely on sophisticated dual-beam systems that involve harmful UV-light and lack specific recognition of biomolecules. Here we develop unique intracellular biomolecule-activatable photoswitches tailored for single-visible-light performed STORM imaging. Upon incorporating intramolecular proton transfer (IPT) units into the photochromic diarylethene, the all-visible-light driven photoswitches are established with excellent photoresponsive efficiency, high brightness and fluorescence ON-to-OFF contrast ratio, guaranteeing STORM imaging using a single-visible-light (488 nm) by regulating the activation, excitation and deactivation processes. Furthermore, we functionalized the IPT units with biomolecular recognition motifs, creating photoswitches capable of sensing the expression levels of intracellular biomolecules (like glutathione (GSH) or β-galactosidase (β-Gal)) with super-resolution. Our objective is to engineer single-visible-light driven, biomolecule-activatable photoswitches, which will significantly streamline the STORM technique and expand the applicability of super-resolution imaging for the precise mapping of intracellular substructures.