Multiscale chemical cartography of single cells: Deconstructing endothelial heterogeneity with synchrotron infrared...

Cellular heterogeneity is a fundamental hallmark of biological systems, yet mapping its biochemical basis quantitatively across from cell populations to subcellular compartments remains a significant challenge. Here, we introduce an integrated analytical framework combining synchrotron infrared microspectroscopy and advanced chemometrics to analyze endothelial heterogeneity at multiple levels. We quantitatively assessed intercellular heterogeneity within a population, mapped intracellular spatial heterogeneity, and decoded the dynamic spatiotemporal responses to lipopolysaccharide (LPS) stimulation. Our analysis revealed that LPS induces a convergent inflammatory state at the population level. Two-dimensional correlation spectroscopy analysis suggested that LPS stimulation initiates a dominant 'outside-in' inflammatory sequence that modifies the baseline 'inside-out' hierarchy. This provides mechanistic insight into the spatiotemporal dynamics and metabolic reprogramming of early inflammatory signaling. Our study establishes a powerful, label-free paradigm for multiscale biochemical imaging that can be applied to the broader study of cellular function and dysfunction in pharmacology, toxicology, and disease pathology.