Fig. 1: Condensate and amyloid formation of FL α-syn under phase separation conditions.

a A diagram outlining the methodology used to monitor α-syn liquid condensate formation, sedimentation and maturation. Samples, loaded in a 96-well glass-bottom plate sealed with a clear film, are incubated at 37°C. Automated 3-step z-stack imaging in solution, alongside bottom-of-well imaging, is performed at selected time points over 20 h. Solution image analysis is then performed to quantify the number and area of all in focus objects within the image. b Representative DIC images acquired in solution during FL α-syn incubation. Scale bars represent 25 μm. c DIC microscopy-derived object area distribution over time. Each timepoint was compiled over three z-stack images. Mean areas are indicated by a solid black line. d Normalized total object count (left Y-axis) and normalized ThT intensity (right Y-axis) as a function of time. Three biological repeats are shown for the normalized total object count data (circles, squares, or triangles), which are globally fitted using a one-phase decay model (black line). Three biological repeats are shown for the normalized ThT aggregation data (blue lines), where each biological repeat is the mean of three technical replicates and error bars represent the standard deviation of the mean. e Representative DIC images acquired at the bottom of the well during FL α-syn incubation. Scale bars represent 25 μm. f Representative TEM image, with magnification, of an aliquot collected at the endpoint of a phase separation ThT aggregation assay. Scale bars represent 2 μm and 500 nm, respectively.