Multimodal Raman and Fluorescence Lifetime Imaging: A New Analytical Approach for Resolving Timing-Dependent...

Endothelial dysfunction involves profound metabolic and biochemical disturbances that disrupt vascular homeostasis, yet temporal recovery patterns remain analytically underexplored. This study presents a high-content analytical platform integrating spontaneous Raman scattering, stimulated Raman scattering, coherent anti-Stokes Raman scattering, and fluorescence lifetime imaging microscopy to quantitatively map molecular alterations associated with endothelial dysfunction in vitro. The multimodal approach enables simultaneous assessment of lipid metabolism, biochemical composition, and cellular bioenergetics with subcellular spatial resolution. Distinct spectral biomarkers were identified, including diagnostic Raman bands at 701, 1748, 2884, and 3011 cm-1, complemented by nonspectral descriptors such as reduced cell volume and accelerated lipid droplet formation. Chemometric analysis utilizing principal component analysis, partial least-squares regression, and semiquantitative lipid profiling revealed a pronounced timing-dependent response to enalaprilat. Preventive treatment was found to preserve metabolic integrity, whereas post-treatment induced only partial restitution and generated a divergent metabolic trajectory. These findings demonstrate that early intervention leads to analytically distinguishable molecular states, underscoring the relevance of temporal profiling in endothelial diagnostics. By establishing a multimodal spectroscopic and chemometric framework, this work highlights the analytical power of integrative imaging for resolving subtle metabolic phenotypes and refining strategies for the objective evaluation of therapeutic efficacy.