Super-resolution imaging of multiple organelle interactions between lysosome-lipid droplets-mitochondria

Unraveling lipid droplets (LDs)-mitochondria-lysosomes crosstalk is vital for decoding cellular states, yet commercial probes suffer from spectral bleed-through, limiting super-resolution imaging. To address this, we innovatively synthesized BDPQ, a LD-targeting fluorescent probe with narrow emission, low toxicity, high cell permeability, and superior photostability-overcoming the flaws of commercial BODIPY dyes. Combining structured illumination microscopy (SIM) with a custom algorithm-assisted analysis, we developed a unique 3D tri-color super-resolution technique, enabling precise quantification of lipophagy, mitophagy, and coordinated lipophagy and mitophagy (named coLipo-Mitophagy) in single cells. Results revealed starvation-induced low-level coLipo-Mitophagy sustains cell viability, while mitochondrial damage drives high-level autophagy and apoptosis. Notably, 2000 kDa hyaluronic acid (HA) was identified as a potential inducer, significantly enhancing coLipo-Mitophagy and triggering LD depletion/apoptosis. This study provides a novel bleed-through-free tool for autophagy-related drug evaluation and inducer discovery.