From Single Molecular Detail to Subcellular Dynamics: Real-Time Kinetics Study of dPA Uptake with Raman-based Spectroscopy

Abstract

Real-time observation of cellular transformations at the subcellular level provides insight into the dynamic biochemical processes underlying cell function and disease. However, many challenges remain in developing methods to analyze metabolic transformations in cells quantitatively. Our study applies Raman spectroscopy (RS) and stimulated Raman scattering (SRS) to link single-molecule resolution with cellular-scale lipid turnover tracking. Using HL-60 cells as a model, we monitor the uptake kinetics of deuterated palmitic acid (dPA), a marker of de novo lipogenesis, from the first minutes to hours. SRS enables microscopic, high-temporal-resolution visualization of dPA incorporation across entire cell volumes, capturing both its distribution and uptake rate. Complementary RS measurements identify distinct metabolic phases: rapid incorporation into pre-existing lipid droplets (LDs) followed by the emergence of new, partially unsaturated LDs associated with cytochrome-linked metabolism. Statistical and spectral analyses reveal cell-to-cell heterogeneity, emphasizing the complexity of lipid metabolism at both the cellular and subcellular levels. By integrating SRS and RS data into time-dependent kinetic profiles, we establish a unified submicron-to-cellular model of fatty acid metabolism. This multi-resolution spectroscopic approach demonstrates how real-time, label-based tracking can uncover dynamic metabolic heterogeneity, advancing understanding of the fatty acid uptake and metabolism in the microscale.