High‐Specificity Spatiotemporal Cholesterol Detection by Quadrature Phase‐Shifted Polarization Stimulated Raman Imaging

Abstract Visualizing cholesterol dynamics in living systems in situ remains a fundamental challenge in biomedical imaging. Although fluorescence microscopy requires bulky tags that perturb small molecule behavior, stimulated Raman scattering (SRS) microscopy enables label‐free detection of CH‐rich molecules. However, conventional SRS probes only polarized Raman components, limiting molecular specificity by seemingly overlapped peaks. Here, we extend SRS microscopy to achieve rapid, comprehensive detection of Raman tensor through quadrature phase‐shifted polarization SRS (QP 2 ‐SRS) microscopy. This technique exploits the underlying molecular signatures by detecting both polarized and depolarized components of third‐order nonlinear susceptibility χ (3) that originates from molecular structural features. We adopt a specialized optical delay line that rapidly alternates between parallel‐ and perpendicular‐polarization states. QP 2 ‐SRS enables unprecedented distinction of similar molecular species in complex mixtures, demonstrating approximately 10× enhancement in chemical specificity and 5× improvement in analytical accuracy. This enhanced sensitivity enables real‐time monitoring of lipid dynamics in living C. elegans and reveals component heterogeneity and morphological changes of LD in NAFLD livers. QP 2 ‐SRS creates new opportunities for investigating cholesterol‐dependent biological processes in their native environment, with broad potential for chemical imaging with enhanced molecular specificity.