Naphthalimide‐Based Dyed Long Fluorescent Lifetime Probe for Lipid Droplet Viscosity Mapping in Non‐Alcoholic Fatty Liver Disease

ABSTRACT Monitoring viscosity dynamics in lipid droplets is crucial for understanding the pathogenesis of non‐alcoholic fatty liver disease (NAFLD), a condition affecting 25% of the global population. However, the application of fluorescence lifetime imaging microscopy (FLIM) for this purpose is hampered by the lack of probes with long fluorescence lifetimes and high signal‐to‐noise ratios. Herein, we developed two naphthalimide‐based dyed fluorescent probes, NBC and NBA, which are viscosity‐sensitive and feature donor‐π‐acceptor architectures. They operate via the restriction of intramolecular rotation in high‐viscosity environments exhibiting 10.7/9.6‐fold fluorescence enhancement with strong linear correlations. In studies with HepG2 cells, both probes demonstrated excellent biocompatibility, specific targeting of lipid droplets, and remarkable photostability during prolonged imaging. FLIM analysis revealed dose‐dependent increases in lipid droplet viscosity following oleic acid treatment. The fluorescence lifetimes increased from 1.3 to 7.0 ns for NBA and from 2.5 to 8.2 ns for NBC. Time‐course experiments using the NBC probe captured distinct lipid droplet maturation stages, showing a progressive viscosity increase from newly formed droplets with a lifetime of 2.2 ns to mature droplets at 7.9 ns. These findings establish NBC and NBA as powerful tools for quantitative lipid droplet viscosity mapping, providing new insights into NAFLD pathogenesis and potentially enabling early diagnostic approaches for lipid metabolism disorders.