JACS Au. 2025 Apr 28. 5(4): 2004-2014
Lysosomes are dynamic, membrane-bound organelles that play key roles in cellular waste disposal, macromolecule recycling, and signaling. Disruptions in lysosomal function and lipid composition are implicated in a wide range of diseases including lysosomal storage disorders, fatty liver disease, atherosclerosis, and cancer. Imaging of the lysosomal lipid composition has the potential to not only enhance the understanding of lysosome-related diseases and their progression but also help identify them. In this work, we present a novel viscosity-sensitive, green-emitting BODIPY probe that can distinguish between ordered and disordered lipid phases and selectively internalize into the lysosomal membranes of live cells. Through the use of fluorescence lifetime imaging microscopy, we demonstrate that lysosomal membranes in multiple cancer cells exhibit significantly higher microviscosities compared to noncancer cells. The differences in lysosomal microviscosities provide an effective approach for identifying cancer cells and indicate that malignant cells may possess more oxidized and saturated lysosomal lipid membranes. Furthermore, we demonstrate the utility of viscosity-sensitive probes in quantifying the compositional changes in lysosomal membranes by investigating the effects of lysosome-permeabilizing cationic amphiphilic drugs (CADs), sertraline, and astemizole. Our results reveal that despite their functional similarities, these CADs exert opposite effects on lysosomal microviscosities in both cancerous and noncancerous cells, suggesting that different mechanisms may contribute to the CAD-induced lysosomal damage and leakage.