Single-Cell Fluorescence Analysis of Lipid Droplet Compositional Dynamics during Triacylglycerol Catabolism

Lipid droplets (LDs) are dynamic organelles essential to lipid metabolism and energy homeostasis, yet their compositional heterogeneity in living cells remains poorly understood. Here, we present LipiPB Red, a red-emissive fluorescent probe with exceptional photostability, high polarity sensitivity, and long-term retention within LDs, even under serum-containing conditions. When applied to fluorescence lifetime imaging microscopy (FLIM), LipiPB Red enables discrimination between triacylglycerol (TAG) and diacylglycerol (DAG) content within individual LDs. FLIM analysis revealed pronounced compositional heterogeneity among LDs in hepatoma cells. This heterogeneity was abolished, with a concomitant increase in average fluorescence lifetimes, upon genetic knockdown or pharmacological inhibition of adipose triglyceride lipase (ATGL), implicating ATGL-mediated lipolysis as a key regulator of LD diversity. Moreover, coimaging with autophagy markers revealed reduced fluorescence lifetimes in LDs localized within both autophagosomes and autolysosomes. These findings indicate a sequential lipid degradation cascade during lipophagy, where lipid hydrolysis is initiated prior to lysosomal fusion.