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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. [ABSTRACT FROM AUTHOR]

Although super‐resolution imaging offers an opportunity to visualize cellular structures and organelles at the nanoscale level, cellular heterogeneity and unpredictability still pose a significant challenge in the dynamic imaging of live cells. It is thus vital to develop better‐performing and more photostable probes for long‐term super‐resolution imaging. Herein, we report a probe, LD‐FG, for imaging lipid droplet (LD) dynamics using structured illumination microscopy (SIM). LD‐FG allows wash‐free imaging of LDs, owing to a hydrogen‐bond sensitive fluorogenic response. The replacement of photobleached LD‐FG by intact probe molecules outside the LDs ensures the long‐time stability of the fluorescence imaging. With this buffering fluorogenic probe, fast and unpredictable dynamic processes of LDs can be visualized. Using this probe, two LD coalescence modes were discovered. The dynamic imaging also allowed us to propose a new model of LD maturation during adipocyte differentiation, i.e. a fast LD coalescence followed by a slow ripening step. The excellent performance of LD‐FG makes the buffer strategy an effective method for designing fluorescent probes for cell dynamic imaging. [ABSTRACT FROM AUTHOR]

Subcellular pH plays a key role to modulate protein structures or specific active sites to impact diverse biological processes. The heterogeneity and high dynamics of intracellular pH require probes that can detect pH in the vicinity of the protein and have a wide pH response range. Here, we reported a protein proximity ligation fluorescent probe for super-resolution imaging of subcellular pH dynamics. Relying on SNAP-tag protein labeling technology, the pH-sensitive fluorophore BG-NDM based on photo-induced electron transfer mechanism was localized to the adjacent positions of the target protein in different subcellular organelles. The equilibrium between the non-fluorescent aggregate and the fluorogenic monomer enabled BG-NDM to have the ability of wash-free super-resolution imaging in live cells. And the wide pH response ranges from pH 6 to pH 12 made the probe have the ability of recognizing pH dynamics. The pH changes around functional proteins during mitophagy, cellular alkalosis and acidosis were revealed. Through long-term super-resolution imaging, we could further record the pH dynamics around mitochondrial proteins COX8A during mitochondrial contacts, fusion and fission at the resolution of a single mitochondria. [Display omitted] • Probe BG-NDM was used to super-resolution imaging of subcellular pH dynamics. • SNAP-tag labeling ensures that BG-NDM detects the pH near the protein of interest. • The aggregate/monomer equilibrium endowed BG-NDM wash-free imaging in living cells. • Mitochondrial pH dynamics was recorded with Long-term SIM imaging. [ABSTRACT FROM AUTHOR]