Nanoscale interrogation of α-Synuclein oligomers: Bridging biophysics, interface science, and neurobiology.

α-Synuclein (α-syn) is an intrinsically disordered presynaptic protein. In synucleinopathies, it undergoes a structural transition into β-sheet-rich conformers that promote the formation of amyloid fibrils and pathological inclusions. Although fibrillar aggregates have been studied extensively, soluble oligomers, which may have the greatest neurotoxic potential, remain poorly understood because of their transient nature and structural heterogeneity. This review critically examines α-syn oligomeric species, with particular emphasis on advanced biophysical and surface characterization techniques and neurobiological models to elucidate oligomer formation, membrane interactions, and toxic mechanisms. Recent advances in spectroscopy, high-resolution microscopy, and mass spectrometry have significantly expanded the ability to characterize α-syn oligomers and their aggregation pathways. Nanopore-based and single-molecule approaches enable the investigation of transient and structurally heterogeneous oligomeric species at the level of individual particles. Interface science has further clarified direct interactions between oligomers and lipid membranes, providing mechanistic insight into neurotoxicity and the lipid-dependent modulation of α-syn conformation and stability. Neurobiological models have revealed multi-organelle disruption, prion-like propagation, and disease subtype-specific seeding. However, no single approach fully captures oligomer pathogenicity, which emerges from the interplay of structure, interfacial behavior, and cellular vulnerability. However, existing frameworks do not adequately address this complexity. Additional barriers include poor reproducibility and limited sensitivity across approaches. Future work should integrate these technologies with standardized biological protocols, advanced artificial intelligence algorithms, and biocompatible nanomaterials. Such an interdisciplinary approach could enable the development of multiscale platforms for real-time studies of α-syn soluble conformers, with clinical utility in the management of synucleinopathies.