Fig. 1.

Detecting aggregate species with ThT, ProteoStat, and AT630. (A) Schematic representation of small aggregate and fibril detection with fluorophores that reversibly bind to aggregates. Unbound fluorophores (in gray) are nonfluorescent. Binding to aggregates locks the fluorophores in a high-fluorescence conformation (in blue), from which aggregate morphology can be detected. (B) Representative TIRF images of aggregates assembled from recombinant αS stained with ThT and ProteoStat (Top) or ThT and AT630 (Bottom). Laser power was set at 10 mW unless otherwise stated. Scale bars represent 5 µm. (C) TIRF images of typical aggregates ranging from 0.2 to 5 µm in size detected with ThT (Top), ProteoStat (Middle), and AT630 (Bottom). Scale bars represent 1 µm. (D) Semilog plot of frequency count of aggregates detected up to 60 µm with each fluorophore. Kolmogorov-Smirnov test indicates that AT630 and Proteostat detected a significantly greater number of aggregates (N_AT630 = 4,417; N_ProteoStat = 4,473; N_ThT = 2,353) and are more faithful at detecting larger aggregates as evidenced by their greater medians and interquartile ranges (IQRs): IQR (median[IQR])_AT630 = 2,428[655–7,260] nm; median[IQR]_ProteoStat = 1,763[452–5,712] nm; median[IQR]_ThT = 1,499[490–4,432] nm; P < 0.0005). See SI Appendix, Fig. S1 for biophysical properties of the three fluorophores.