Structural diversity of Alzheimer-related protein aggregations revealed using photothermal ratio-metric micro-spectroscopy

The crucial link between pathological protein aggregations and lipids in Alzheimer's disease pathogenesis is increasingly recognized, yet its spatial dynamics remain challenging for labeling-based microscopy. Here, we demonstrate photothermal ratio-metric infrared spectro-microscopy (PRISM) to investigate the <i>in situ</i> structural and molecular compositions of pathological features in brain tissues at submicron resolution. By identifying the vibrational spectroscopic signatures of protein secondary structures and lipids, PRISM tracks the structural dynamics of pathological proteins, including amyloid and hyperphosphorylated Tau (pTau). Amyloid-associated lipid features in major brain regions were observed, notably the enrichment of lipid-dissociated plaques in the hippocampus. Spectroscopic profiling of pTau revealed significant heterogeneity in phosphorylation levels and a distinct lipid-pTau relationship that contrasts with the anticipated lipid-plaque correlation. Beyond <i>in vitro</i> studies, our findings provide direct visualization evidence of aggregate-lipid interactions across the brain, offering new insights into mechanistic and therapeutic research of neurodegenerative diseases.