Analyzing the Cholesteryl Ester Fraction in Lipid Droplets with a Polarity-Ultrasensitive Fluorescence Lifetime Probe

Quantifying the lipid composition of cellular lipid droplets (LDs) in situ is challenging but crucial for understanding lipid metabolic diseases. Here, we propose a fluorescence lifetime imaging method based on a polarity-sensitive probe (LD660) for analyzing the lipid composition of the LDs. The probe emits strong fluorescence at 660 nm only in apolar LD environments, with dielectric constants of 2–4, and outperforms Nile red in LD imaging. Importantly, the fluorescence lifetime of LD660 increases with the incremental fraction of cholesteryl ester in neutral lipid mixtures. Using fluorescence lifetime microscopy with LD660, we imaged and quantified the cholesteryl ester fractions of LDs in cells and tissues. It is found that macrophages and surrounding hepatocytes in fatty liver diseases show significantly higher cholesteryl ester contents than other hepatocytes. This finding suggests that cholesteryl ester may serve as a potential indicator of the degree of hepatic steatosis.