Lipid profiling at submicron resolution by hyperspectral stimulated Raman imaging

Achieving high spatial resolution in lipid profiling for life science and biomedical applications remains challenging. Despite their structural similarities, lipids play specialized cellular roles, requiring methods capable of subcellular compositional analysis. Conventional techniques based on lipid extraction lack the spatial resolution needed for subcellular tracking. Vibrational spectroscopy, targeting chemical bonds such as C-H and C=C, offers a promising alternative. Here, we employed hyperspectral stimulated Raman scattering (SRS) imaging to profile lipids in single cells by identifying their spectroscopic signatures. Imaging two types of bladder cancer cells revealed the accumulation of unsaturated lipids in lipid droplets. Furthermore, simultaneous two-photon excitation fluorescence imaging and biomolecular spectroscopic analysis revealed micronucleus formation, a marker of genome instability, which are found to be surrounded by lipid droplets with high unsaturation level. These results demonstrate the application of hyperspectral SRS imaging for submicron spatially resolved lipid profiling for providing new insight in cancer aggressiveness mechanisms.