Revealing Alkali Ion Modulation on the Nucleation Phase of Highly Concentrated α-Synuclein Probed by Thioflavin T Lasing Spectroscopy.

Abstract Early nucleation events during α-synuclein (αS) aggregation remain challenging to investigate because conventional fluorescence assays lose sensitivity in highly concentrated biomolecular environments. Here, we present Thioflavin T (ThT) lasing spectroscopy in a Fabry-Pérot microcavity as a sensitive methodological approach for probing αS aggregation within condensate-like microenvironments that more closely mimic intracellular molecular crowding. Beyond conventional lasing-threshold analysis, we introduce the first automated frame-by-frame analysis of ThT lasing spectra using machine-vision algorithms, enabling statistical evaluation of individual lasing events throughout the aggregation process. As a demonstration of this methodology, we investigate how physiologically abundant monovalent and divalent cations (Na+, K+, Mg2+, and Ca2+) modulate the nucleation phase of highly concentrated αS. Ion-specific differences were identified in both lasing-threshold kinetics and spectral evolution. Monovalent cations produced complex, non-monotonic threshold behavior accompanied by transient spectral shifts, consistent with incomplete electrostatic screening and dynamic accessibility of ThT to early oligomeric species. In contrast, divalent cations generated more stable lasing wavelengths and distinct threshold regimes, reflecting stronger electrostatic screening. These results demonstrate that automated analysis of ThT lasing provides complementary information beyond conventional fluorescence, enabling sensitive interrogation of nucleation processes in highly crowded protein systems. The presented methodology establishes lasing spectroscopy combined with machine-vision analysis as a powerful platform for investigating early protein aggregation under physiologically relevant condensed conditions.