Stable super-resolution imaging of lipid droplet dynamics through a buffer strategy with a hydrogen-bond sensitive fluorogenic probe.

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 hydrogen-bond sensitive fluorogenicity. The replenishment of photobleached LD-FG by intact ones outside LDs further ensure the long-time stability of the fluorescence imaging. With this buffering fluorogenic probe, fast and unpredictable dynamic processes of LDs can be visualized. Two LD coalescence modes (as well as heterogeneity in different regions of the cells and even in between different cells) were discovered for the first time. Notably, 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.