Exploring the potential for scanning electron microscopy/focused ion beam - based diffraction for screening cryo-transmission electron microscopy samples

Abstract The study of biological and organic materials at high resolution using cryogenic transmission-electron microscopy (cryo-TEM) necessitates vitrification to preserve the native structure. Assessing sample integrity is essential, particularly as ice crystallization during freezing and handling can cause irrecoverable structural damage. Usually, a secondary cryo-TEM is used for initial screening, only possible after a time-consuming sample preparation workflow. In the present work, we propose simple methods that exploit existing workflows developed for materials science analyses and demonstrate on-grid in situ assessment of ice crystallinity with electron backscatter diffraction (EBSD) on a direct electron detector (DED) in a cryo-scanning-electron microscope (SEM). This evaluation step can be performed prior to sample preparation for cryo-TEM by using cryogenic focused ion beam (cryo-FIB) milling. Custom grid holders and jigs were developed to integrate the clipped cryo-TEM grids and evolve the sample preparation workflow. EBSD detects hexagonal ice in some areas of the samples, whereas other areas show an absence of EBSD signal, consistent with vitreous ice, that enable targeting the further steps of sample preparation for cryo-TEM. Off-axis transmission Kikuchi diffraction (TKD) was attempted, but led to severe damage to polished TEM-lamellae and appears unsuitable. A proof-of-concept lift-out from a clipped cryo-TEM grid mounted on a support is introduced, demonstrating possibilities for expanded cryogenic correlative workflows beyond the acceleration of sample screening for cryo-TEM. Competing Interest Statement C.W. provides cryo-EM consulting services. R.D.K. is employed by EDAX / Gatan, a subsidiary of Ametek. Footnotes new figure added to SI, changed figure in main text, corrected 3 details in methods