Real-Time Analysis of Lipid Droplet Morpho-Chemical\nDynamics in Living Human Hepatocytes via Phase-Guided Raman Sampling

Lipid\ndroplets (LDs) are highly dynamic organelles, undertaking\nmany important functions such as maintaining lipid metabolism and\ncellular homeostasis. Traditional methods to analyze LD dynamics focus\non morphological changes, while chemical dynamics cannot be easily\nprobed with traditional analytical chemistry techniques. To overcome\nthis challenge, we show here how our phase-guided Raman sampling method,\nwhere high-resolution phase microscopy images direct a Raman sampling\nbeam, can perform label-free, multimodal characterization of LD dynamics\nin living cells at both the single-cell and single-LD levels with\nsubmicron accuracy and high temporal resolution. We demonstrate the\nstudy of the morphological–compositional dynamics of human\nhepatocellular carcinoma cells (PLC cells) under different environmental\nconditions and with and without fatty acid supplementation, providing\ninsight into LD heterogeneity and heterogeneity of response. Finally,\nwe introduce a measurement method for the dynamics of cell-average\nLD composition, which can quickly and accurately characterize the\nlipid dynamics at the single-cell level with <30 s temporal resolution.\nThe results here show the promise of the phase-guided Raman sampling\nmethod for dynamic morpho-chemical profiling of organelle populations.