Visualization of Lipid Droplets in Living Cells Using (4‐Methoxyphenyl) Ethynyl‐Naphthalimide

Fluorescence sensors provide powerful platforms for real‐time monitoring of viscosity due to their high sensitivity, with important implications for microscopic and cellular studies. Lipid droplets, which play central roles in cellular metabolism, represent key targets for such investigations; however, precise approaches for quantifying intracellular viscosity and polarity remain limited. To address this challenge, we developed a series of (4‐methoxyphenyl) ethynyl‐naphthalimide derivatives tailored for viscosity sensing and live‐cell imaging. These fluorophores displayed pronounced fluorescence enhancement, with a linear emission response in glycerol solutions ranging from 60% to 100%, demonstrating high sensitivity to viscosity changes. In addition to favorable photophysical properties, they exhibited excellent biocompatibility and selective accumulation in lipid droplets, enabling real‐time visualization of viscosity dynamics in live cells. Among these, probe LD‐A showed superior performance: medium‐viscosity environments correlated with an increased green‐to‐blue fluorescence intensity ratio, while high‐viscosity conditions induced redshifted emissions, consistent with in vitro solvent studies. Collectively, these results establish LD‐A and its analogs as promising fluorescent tools for probing viscosity variations in biological systems with high specificity and sensitivity.