IR-AMES enables label-free vibrational fingerprinting of individual biomolecular assemblies under native aqueous conditions and reveals that Alzheimer's disease-derived tau oligomers are distinguished by enrichment of antiparallel β-sheet structures and RNA components that drive enhanced neurotoxicity through preferential interactions with anionic membranes accompanied by structural destabilization.

Tau misfolding and aggregation are central to cognitive decline in Alzheimer’s disease and related neurodegenerative disorders. Although soluble tau oligomers are implicated as primary toxic species, the structural and compositional determinants of their toxicity remain inaccessible at the single oligomer level. Here we introduce infrared absorbance-modulated evanescent scattering (IR-AMES), a label-free single-molecule spectroscopic imaging approach that photothermally encodes mid-infrared vibrational fingerprints into evanescent scattering from individual biomolecular assemblies under native aqueous conditions. Applying IR-AMES to recombinant human tau resolves random-coil-dominated monomers and captures the emergence of structurally heterogeneous oligomers. Analysis of tau oligomers from postmortem Alzheimer’s disease brains uncovers enrichment of antiparallel β-sheet structures and RNA components, features that are largely obscured in ensemble-averaged measurements. Using lipid nanodiscs as a defined membrane mimic, we further show that pathological tau oligomers exhibit enhanced interactions with anionic membranes. Together, these findings establish a link between structure and neurotoxicity of tau oligomers, and position IR-AMES as a platform for uncovering structure–function relationships in complex biomolecular assemblies.