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bioRxiv · 10.1101/2023.04.28.538734

Serial Lift-Out - Sampling the Molecular Anatomy of Whole Organisms

Schioetz, O. H.·Kaiser, C. J. O.·Klumpe, S.·Morado, D. R.·Poege, M.·Schneider, J.·Beck, F.·Thompson, C.·Plitzko, J. M.
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Abstract

Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically < 300 nm. Here, we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to C. elegans L1 larvae yielding a cryo-ET dataset sampling the worms anterior-posterior axis and resolve its ribosome structure to 7 [A], illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.

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Schioetz, O. H., Kaiser, C. J. O., Klumpe, S., Morado, D. R., Poege, M., Schneider, J., Beck, F., Thompson, C., Plitzko, J. M.. 2023-04-30. Serial Lift-Out - Sampling the Molecular Anatomy of Whole Organisms. https://doi.org/10.1101/2023.04.28.538734

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