2100 cryo tem

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.

Liquid-phase TEM imaging was carried out on a JEOL 2100 Cryo TEM with a spot size 3 with a LaB₆ emitter at 200 kV using the Protochips Poseidon 210 liquid flow holder. The illumination area is intentionally kept larger than the fluorescent screen of the TEM (~35 cm). The electron beam is sufficiently spread out to minimize artefacts from the boundary and nonuniformity of the illumination area on the supracrystal growth and to achieve sufficiently low dose rates. The movies were captured by a Gatan Ultrascan charge-coupled device (CCD) camera with a 0.1 s exposure time per frame at a rate of 1.3 frames per seconds (fps). In a typical experiment, an aliquot of the nanoprism solution prepared above (34.5 mM pH = 8 PBS buffer solution) was micropipetted on a SiN_x chip (window: 550 μm × 20 μm, 150 nm spacer flow echip, Protochips), which was then assembled with another SiN_x chip (window: 550 μm × 20 μm) in a Protochips Poseidon 210 liquid flow TEM holder. The SiN_x chips were pretreated at a medium RF level for 45 s using a Harrick PDC-23G basic plasma cleaner to render them clean and hydrophilic. During the liquid-phase TEM imaging, the electron dose rates were kept low (3.7–14.8 e^– Å^–2 s^–1). At this dose rate, the thiol ligands on the nanoprism have been shown to stay intact on the particle surface and remain negatively charged in our previous studies^{31 (link),32 (link),45 (link)}.