Usc f0.8 k0 1 t12

High-speed AFM (HS-AFM) imaging was performed using tip scan high-speed AFM (BIXAM, Olympus, Tokyo, Japan), which was improved based on a developed prototype AFM^{43 (link)}. A 2 μL drop of the 0.5 to 1 nM sample in buffer composed of 5 mM Tris-HCl (pH 8.0), 15 mM MgCl₂, 1 mM EDTA with or without 100 mM KCl was deposited onto a freshly cleaved mica surface (diameter 3.0 mm) and incubated for 1 min. The surface was subsequently rinsed with 10 µL of the same buffer and then scanned in ≈120 μL of the buffer containing designated concentrations of KCl. Small cantilevers (9 µm long, 2 µm wide, and 100 nm thick) with an electron-beam-deposited carbon tip (tip length ≈2 μm, tip radius <10 nm) having a spring constant of 0.1 N m⁻¹ and a resonant frequency of ≈300–600 kHz in water (USC-F0.8-k0.1-T12; Nanoworld, Neuchâtel, Switzerland) were used to scan the sample surface. The 320 × 240-pixel images were collected at a scan rate of 0.5 frames per second with tapping mode. The images were analyzed using AFM scanning software (Olympus) and ImageJ software (http: //imagej.nih.gov/ij/).

For imaging the aggregation of Aβ42, 20-µL samples of Aβ42 (10 µM in PBS) and 10-µL lipid (DOPC, DOPC/Chol., DOPC/SM/Chol., or BTLE) were incubated in Protein LoBind Tubes for 2 h at room temperature. Two-microliter samples of Aβ42 solution were deposited onto a freshly cleaved mica surface. After incubation for 10 min at room temperature, the mica surface was gently washed with PBS buffer. The AFM imaging of Aβ42 aggregations was performed in PBS buffer using a high-speed AFM system (BIXAM, Olympus, Tokyo, Japan) with a silicon nitride cantilever (resonant frequency = 0.8 MHz, spring constant = 0.1 N/m, EBD tip radius < 10 nm; USC-F0.8-k0.1-T12; Nanoworld, Neuchâtel, Switzerland) (35 (link)). The images of 320 × 240 pixels were obtained at a scan rate of 0.2 fps for static images.