Isolation of a Ubiquitous Cholesteryl Ester Spectral Signature.

Lipid droplet (LD) composition is central to metabolic regulation and disease progression, yet its quantitative analysis is hindered by substantial spectral overlap between triacylglycerols (TAGs) and cholesteryl esters (CEs), which share similar acyl chain structures. Here, using stimulated Raman scattering (SRS), we isolate a ubiquitous CE spectral signature that is conserved across CE species and independent of acyl chain identity. This previously hidden "CE-core" spectrum contains features with minimal overlap with TAG spectra, enabling CE-specific differentiation. We extract the CE-core spectrum using gradient-based residual iterative minimization (GRIM), a spectral unmixing framework developed in this study. Using this conserved signature, we quantify TAG-to-CE ratios across varied cellular conditions and resolve LD heterogeneity associated with differential fatty acid incorporation. These findings demonstrate that conserved molecular signatures can be uncovered within overlapping spectral regimes to enable chemical insight and quantitative analysis in complex biological systems.