All AFM imaging was performed in liquid using Peakforce Tapping with either a Dimension FastScan microscope (images presented in Figs 1A and B , and 2A and D, and EV1 ) or a Multimode 8 (images presented in Fig 1C ). FastScan D cantilevers (Bruker) were used with the Dimension FastScan microscope and PeakForce HIRS‐F‐B cantilevers (Bruker) were used with the Multimode 8 microscope. FastScan D cantilevers were operated using a drive frequency of 8 kHz and PeakForce HIRS‐F‐B cantilevers were operated at 4 kHz drive frequency. The areas imaged were 0.8 × 0.25–4 × 2 µm2 in size and recorded at 3.5 Hz (FastScan) or 1.75 Hz (Multimode 8) line rate. Images were 512 × 256, 512 × 172 or 384 × 172 pixels in size.
During AFM movie acquisition, disaggregation was initiated by retracting the cantilever tip ~100 nm from the surface and injecting 0.75–1 µM Hsc70, 0.37 – 0.5 µM DNAJB1 and 0.07–0.1 µM Apg2 (always at a Hsc70:DNAJB1:Apg2 molar ratio of 1:0.5:0.1) in disaggregation buffer (HKMD buffer for ‐ATP controls) into the sample droplet. AFM imaging was continued within 1 min of injection of chaperones. The same area of αSyn fibres was imaged for >2 h at either at room temperature or at 30°C.
During AFM movie acquisition, disaggregation was initiated by retracting the cantilever tip ~100 nm from the surface and injecting 0.75–1 µM Hsc70, 0.37 – 0.5 µM DNAJB1 and 0.07–0.1 µM Apg2 (always at a Hsc70:DNAJB1:Apg2 molar ratio of 1:0.5:0.1) in disaggregation buffer (HKMD buffer for ‐ATP controls) into the sample droplet. AFM imaging was continued within 1 min of injection of chaperones. The same area of αSyn fibres was imaged for >2 h at either at room temperature or at 30°C.