TEM Imaging of Amyloid Fibrils

TEM grids were prepared using a 1 min oxygen plasma cleaning treatment (Harrick Plasma Cleaner PDC-32G, at low power). One 10 µl droplet of Asyn fibril suspension and three 10 µl droplets of ultrapure water for each grid were pipetted onto a clean sheet of Parafilm. Freshly plasma-cleaned TEM grids were inverted onto a sample droplet and rested for 60 s. Excess sample solution was blotted away with filter paper, and grids were placed onto each of the three droplets of water and blotted again in quick succession, to rinse away excess salt. Grids were then rested on a droplet of tobacco mosaic virus (TMV) suspension, prepared by diluting a stock solution to 0.12 mg/mL. TMV was used to calibrate electron density in each image^{93 (link)–95 (link)}. Imaging was done on a JEOL 2100 Cryo-TEM using an electron accelerating voltage of 80 kV. Micrographs were collected in the tilt-beam geometry using the third objective aperture, an exposure time of 3–5 s. Short, non-overlapping segments of TMV and Asyn fibrils in each image were then selected using the helixboxer function of EMAN2^{96 (link)} and exported to the MpUL-multi program for quantification of MPL statistics^{97 (link)}. Segments were only chosen from TMV and Asyn fibrils when they were distinguishable in both tilt-beam and accompanying bright-field micrographs.