Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA.
Methods Mol Biol. 2021;2215:125-144. doi: 10.1007/978-1-0716-0966-8_6.
Illuminating a specimen with a parallel electron beam is critical for many experiments in transmission electron microscopy as deviations from this condition cause considerable deterioration of image quality. Carefully establishing parallel illumination is particularly important on two-condenser lens transmission electron microscopes (TEMs) as the parallel illumination condition is limited to a single beam intensity value on these instruments. It was recently shown that a Thermo Fisher Scientific Talos Arctica, a two-condenser lens TEM operating at 200 kV, equipped with a Gatan K2 Summit direct electron detector is capable of resolving frozen-hydrated macromolecules of various sizes and internal symmetries to better than 3 Å resolution using single particle methodologies. A critical aspect of the success of these findings was the careful alignment of the electron microscope to ensure the specimen was illuminated with a parallel electron beam. Here, this chapter describes how to establish parallel illumination conditions in a Talos Arctica TEM for high-resolution cryogenic data collection for structure determination.
用平行电子束照射样品对于透射电子显微镜中的许多实验至关重要，因为偏离这种条件会导致图像质量的严重恶化。在双聚光镜透射电子显微镜（TEM）上，仔细建立平行照明尤为重要，因为在这些仪器上，平行照明条件仅限于单个光束强度值。最近表明，配备 Gatan K2 Summit 直接电子探测器的 Thermo Fisher Scientific Talos Arctica 型 200kV 双聚光镜 TEM 能够使用单颗粒方法解析各种大小和内部对称性的冷冻水合大分子，分辨率优于 3Å。这些发现成功的一个关键方面是仔细调整电子显微镜，以确保样品被平行电子束照射。在这里，本章描述了如何在 Talos Arctica TEM 中建立平行照明条件，以进行高分辨率低温数据收集，用于结构测定。
