Super-resolution imaging, especially a single-molecule localization approach, has raised a fluorophore engineering revolution chasing sparse single-molecule dark-bright blinking transforms. Yet, it is a challenge to structurally devise fluorophores manipulating the single-molecule blinking kinetics. In this pursuit, we have developed a triggering strategy by innovatively integrating the photoactivatable nitroso-caging strategy into self-blinking sulfonamide to form a nitroso-caged sulfonamide rhodamine (NOSR). Our fluorophore demonstrated controllable self-blinking events upon phototriggered caging unit release. This exceptional blink kinetics improved the super-resolution imaging integrity on microtubules compared to self-blinking analogues. With the aid of paramount single-molecule fluorescence kinetics, we successfully reconstructed the ring structure of nuclear pores and the axial morphology of mitochondrial outer membranes. We foresee that our synthetic approach of photoactivation and self-blinking would facilitate rhodamine devising for super-resolution imaging.

Long-term super-resolution imaging appears to be increasingly important for unraveling organelle dynamics at the nanoscale, but is challenging due to the need for highly photostable and environment-sensitive fluorescent probes. Here, we report a self-blinking fluorophore that achieved 12 nm spatial resolution and 20 ms time resolution under acidic lysosomal conditions. This fluorophore was successfully applied in super-resolution imaging of lysosomal dynamics over 40 min. The pH dependence of the dye during blinking made the fluorophore sensitive to lysosomal pH. This probe enables simultaneous dynamic and pH recognition of all lysosomes in the entire cell at the single-lysosome-resolved level, which allowed us to resolve whole-cell lysosome subpopulations based on lysosomal distribution, size, and luminal pH. We also observed a variety of lysosome movement trajectories and different types of interactions modes between lysosomes.