Rapid intracellular detection and analysis of lipid droplets' morpho-chemical composition by phase-guided Raman sampling

Lipid droplets (LDs) are dynamic, complex organelles involved in nearly all aspects of cellular metabolism. In situ characterization methods are primarily limited to fluorescence imaging, which yields limited chemical information, or Raman spectroscopy, which provides excellent chemical profiling but very low throughput. Here we propose a new paradigm where locations of both large and small droplets are obtained automatically from high resolution phase images and fed into a galvo-mirror-controlled Raman sampling arm to obtain the full spectrum of each LD efficiently. Using this phase-guided Raman sampling, we can characterize hundreds of LDs within a single cell in minutes and easily acquired more than 40,000 high quality spectra. The dataset revealed strong, cell-line dependent, cell-dependent, and individual-droplet-dependent composition and saturation level changes to various culture conditions. We further revealed a strong competitive relationship between mono- and poly-unsaturated fatty acids, where supplementation with one led to a relative decrease in the other.