File S1 - Synergistic Interactions between Alzheimer’s Aβ40 and Aβ42 on the Surface of Primary Neurons Revealed by Single Molecule Microscopy

Supporting information. Figure S1. Sample with Aβ40-HL555 shows shorter fluorescence lifetime spots than the control sample. The raw FLIM data is shown on the left and the calculated lifetime image was fitted with single exponential decay. The fluorescent spots were selected based on the fluorescence image and their lifetimes were collected and plotted as shown on the right. Their lifetime distributions were normalized to total number of spots. The lifetime of Aβ40-HL555 peaks at 0.48 ns and is 6 fold more abundant than the autofluorescence, whereas autofluorescence peaks at 0.58 ns. Therefore we conclude any spot with lifetime longer than 0.53 ns is autofluorescence and excluded. The data presented for each sample is the average of two experiments and each experiment contained at least 250 particles. Error bars represent the standard deviation of the mean. Figure S2. FRET is only detected when Aβ40 is mixed with Aβ42. Primary hippocampal neurons incubated with 2nM Aβ40 were excited by 532 nm laser and show Aβ40 (donor) emission (A) but do not show any emission in Aβ42 (acceptor) channel (B), and Aβ40 can not be directly excited by 635 nm (C). Neurons incubated with 2nM Aβ42 only were also excited by 532 nm laser but do not show any signal in Aβ40 (donor) and Aβ42 (acceptor) emission channels (D and E). The sample with just Aβ42 can only be excited by 635 nm laser and shows emission in Aβ42 (acceptor) emission (F). Neurons incubated with 2nM Aβ40 and 2nM Aβ42 were excited by 532 nm laser and show both donor emission (G) and FRET signal (H). Excitation of 635 nm laser confirmed Aβ42 emission co-localizes with acceptor signals (I). The dashed circle shown in (D) indicates the autofluorescence generated by 532 nm laser, and the donor emission is later distinguished from autofluorescence by their fluorescence lifetime. Scale bars are 10 µm. (DOC)