Monitoring the synthesis of neutral lipids in lipid droplets of living human cancer cells using two-color infrared photothermal microscopy

There has been growing interest in the functions of lipid droplets (LDs) due to recent discoveries regarding their diverse roles. These functions encompass lipid metabolism, regulation of lipotoxicity, and signaling pathways that extend beyond their traditional role in energy storage. Consequently, there is a need to examine the molecular dynamics of LDs at the subcellular level. Two-color infrared photothermal microscopy (2C-IPM) has proven to be a valuable tool for elucidating the molecular dynamics occurring in LDs with sub-micrometer spatial resolution and molecular specificity. In this study, we employed the 2C-IPM to investigate the molecular dynamics of LDs in both fixed and living human cancer cells (U2OS cells) using the isotope labeling method. We investigated the synthesis of neutral lipids occurring in individual LDs over time after exposing the cells to excess saturated fatty acids while simultaneously comparing inherent lipid contents in LDs. We anticipate that these research findings will reveal new opportunities for studying lesser-known biological processes within LDs and other subcellular organelles. Two-color infrared photothermal microscopy (2C-IPM) can be used to study lipid metabolism in lipid droplets (LDs). Using deuterated fatty acids, it distinguishes endogenous and newly synthesized lipids, allowing quantitative analysis and monitoring of neutral lipid synthesis in living cell LDs.