Fig. 2. Application to the detection of protein aggregates.
a ThT binds specifically to amyloid fibrils and increases fluorescence upon binding. b Example traces of ThT (1 μm) binding to amyloid fibrils and measurement of different concentrations of mature α-syn amyloid fibrils (405 nm excitation, acquired at 100 Hz for 400 s). c Total ThT intensity detected by AttoBright (light blue) or a bulk measurement using a fluorescence plate reader (BMG Labtech, red). For concentrations below 3 nm (dotted line), the AttoBright data are analysed by integrating individual peaks. For concentrations above 3 nm, the AttoBright data are analysed by integrating total signal intensity. The dotted line marks concentration at which individual fluorescent bursts cannot be reliably separated. The dotted line is three orders of magnitude lower than the typical sensitivity of a bulk measurement. d, e Deconvolution of number and size of aggregates measured in c. d Number of ThT-positive species detected by AttoBright across serial fibril dilutions. Linear range ~ 10−11–10−9 m. e Average peak intensity remains constant with fibril length across a wide range of concentrations (~ 10−11–10-8 m). Error bars are mean ± s.d of 3 × 400 s measurements.