Gradual labeling with fluorogenic probes: A general method for MINFLUX imaging and tracking.

Minimal photon fluxes (MINFLUX) nanoscopy excels in nanoscale protein studies but lacks a universal method for simultaneous imaging and live-cell tracking in dense cellular environments. Here, we developed a general strategy, gradual labeling with fluorogenic probes for MINFLUX (GLF-MINFLUX) imaging and tracking. In GLF-MINFLUX, membrane-permeable small-molecule fluorogenic dye with protein-induced "off/on" switching is gradually labeled, located, and bleached, enabling sequential positioning and tracking of individual proteins. GLF-MINFLUX reveals continuous microtubules with 2.6-nanometer localization precision, offering substantially improved precision (1.7-fold), acquisition (2.2-fold), and target density (3-fold) compared to conventional MINFLUX with Alexa Fluor 647. GLF-MINFLUX also enabled the three-dimensional localization of translocase of the outer mitochondrial membrane 20 proteins within mitochondrial clusters and dual-channel nanoscale imaging of endogenous neuronal microtubules and microfilaments. GLF-MINFLUX allowed live-cell single-protein tracking with 7.8-nanometer precision at ~200-microsecond temporal resolution, revealing distinct diffusion behaviors and rates between the basal membrane and filopodia. GLF-MINFLUX, requiring only tuning of probe concentration, offers molecular-level insights into protein functions.