Single-Molecule Imaging Reveals Differential Stability of Alpha-Synuclein Aggregates

Alpha-Synuclein (-syn) aggregation is central to Parkinson's disease (PD), yet measurements in biofluids are confounded by the coexistence of monomeric and aggregated species. Using Syn-IMAGR, a single-molecule imaging platform with sub-femtomolar sensitivity, we show that purified -syn aggregates undergo dilution-induced disassembly, revealing a concentration-dependent equilibrium. Applied to postmortem brain lysates, Syn-IMAGR distinguishes physiological -syn multimers, which are dimmer and readily dissociate upon dilution, from PD-associated aggregates, which remain detectable and exhibit greater structural resistance to disruption. These results indicate that -syn assemblies occupy distinct stability regimes, with PD-associated aggregates representing a more persistent and less dilution-sensitive structural state. Syn-IMAGR thus provides a quantitative framework for resolving -syn species and for probing their concentration-dependent equilibrium.