Multimode nanoscope iiia microscope

10 μL purified αSOs were added to freshly cleaved mica and allowed to adsorb for 5 min at RT. Unbound protein was gently washed off with Millipore-filtered water (3x 50 μL), dried under a gentle stream of nitrogen and imaged immediately on a MultiMode Nanoscope IIIa microscope (Digital Instruments, USA) equipped with an E-scanner. All measurements were carried out in tapping mode under ambient conditions using single-beam silicon cantilever probes with a resonance frequency of 300 kHz (Olympus, Japan). AFM images were captured with a resolution of 512 samples/line at a scan rate of 1 Hz, with scan sizes of 2x2 μm. Raw data images were processed using Gwyddion software (V2.37) by levelling the data and shifting minimum data values to zero. To determine αSO heights, ~150 αSOs from three different regions were analysed.

Labeled α-syn fibrillar samples were diluted 5 times into PBS, and 10 μL were pipetted onto freshly cleaved mica and kept at room temperature for 60 s. The mica surface was then rinsed with Millipore-filtered water (2×50 03BCL) to remove loosely bound protein, dried under a stream of nitrogen and imaged immediately. AFM imaging was performed on a MultiMode Nanoscope IIIa microscope (Digital Instruments, USA) equipped with an E-scanner. All measurements were carried out in the tapping mode under ambient conditions using single-beam silicon cantilever probes with a resonant frequency of 300 kHz (Olympus, Japan). Image analysis was performed using the instrument software.