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bioRxiv · 10.1101/2022.10.03.510728

CryoFIB milling large tissue samples for cryo-electron tomography

Li, X.·Wang, S.·Zhou, H.·Chen, W.·Jiang, Y.·Yan, X.·You, H.
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Abstract

Cryo-electron tomography (cryoET), a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In particular, locating and cutting out objects of interest from a large tissue sample is an important but difficult step. In this study, we report a sample thinning strategy and workflow for tissue samples based on cryo-focused ion beam (cryoFIB) milling. This workflow provides a full solution for isolating objects of interest by starting from a millimeter-sized tissue sample and ending with hundred-nanometer thin lamellae. The workflow involves sample fixation, pre-sectioning, a two-step milling strategy, and locating the object of interest using cellular secondary electron imaging (CSEI). The two-step milling strategy introduces a coarse milling method to solve the milling efficiency problem for samples as thick as tens of microns, followed by a fine milling method to create a furrow-ridge structure. The furrow-ridge structure guarantees the generation of large, thin lamellae with enhanced mechanical stability and charge-reducing design. CSEI is highlighted in the workflow, which provides conventional, on-the-fly locating during cryoFIB milling. Tests of the complete workflow were conducted to demonstrate the high efficiency and high feasibility of the proposed method.

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Li, X., Wang, S., Zhou, H., Chen, W., Jiang, Y., Yan, X., You, H.. 2022-10-05. CryoFIB milling large tissue samples for cryo-electron tomography. https://doi.org/10.1101/2022.10.03.510728

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