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Figure 3
Sample preparation for macromolecular MicroED. (a) Several examples of crystallization conditions with protein microcrystals of different morphologies suitable for MicroED. Crystals can be identified using light microscopy as shown in the top row, corresponding images of negatively stained samples are shown on the bottom row. Scale bars for the top row are 500 nm and bottom row 400 nm. Images were adapted from the work by Shi et al. (2016BB96). (b) Typical workflow involved in MicroED sample preparation for protein crystals. The crystals are pipetted from the crystallization drop and deposited onto the carbon-side of a glow-discharged EM-grid held between the tips of a tweezer. The crystals are allowed to settle on the grid before any excess liquid is blotted away from the back-side of the grid with filter paper. The grid is then rapidly plunged into liquid ethane for freezing and kept at cryogenic temperatures until use. The grid is either transferred directly to the TEM for MicroED data collection, or can be thinned by cryo-FIB milling into crystalline lamellae suitable for MicroED. (c) FIB milling of tetragonal lysozyme crystals, showing the SEM images (left) side by side with the FIB images (right) during a typical milling workflow as described previously by Martynowycz & Gonen (2021aBB70). After a suitable crystal is identified, rectangular boxes (blue) are drawn for coarse milling the bulk material (top). The resulting 3 µm-thick lamella is then further thinned by polishing using a lower current and smaller step sizes (middle). This results in a thin crystalline lamella of ideally 200–300 nm thickness and a width of 5 µm (bottom). Scale bars for the SEM images from top to bottom are 50, 25 and 25 µm, for the FIB images from top to bottom 10, 10 and 1 µm.

IUCrJ
Volume 9| Part 2| March 2022| Pages 169-179
ISSN: 2052-2525
https://doi.org/10.1107/S2052252521013063