Advances in Probing Amyloid Heterogeneity Using Vibrational Spectroscopy and Imaging

The misfolding and aggregation of proteins into amyloid fibrils are associated with numerous human diseases; however, our understanding of the mechanisms by which amyloid proteins exert their toxicity remains limited. This gap in knowledge can largely be attributed to the significant polymorphism of species that form during aggregation, ranging from short-lived soluble oligomers to various polymorphic fibrils, compounded by the complex interplay of other proteins and biomolecules. Vibrational spectroscopies are particularly well-suited for studying these heterogeneous mixtures and, with the integration of site-specific probes, can provide residue-level structural information. This perspective highlights recent advances in the application of Raman and infrared (IR) spectroscopy and imaging techniques to elucidate aggregation mechanisms, characterize oligomer and fibril structures, analyze plaque compositions, and investigate the effects of coassembly and cross-seeding. These efforts move us toward a greater understanding of how amyloids form under disease-relevant conditions, which may provide new routes toward targeted therapeutics.