We demonstrate gas cluster ion beam scanning electron microscopy (SEM), in which wide-area ion milling is performed on a series of thick tissue sections. This three-dimensional electron microscopy technique acquires datasets with <10 nm isotropic resolution of each section, and these can then be stitched together to span the sectioned volume. Incorporating gas cluster ion beam SEM into existing single-beam and multibeam SEM workflows should be straightforward, increasing reliability while improving z resolution by a factor of three or more.
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The GCIB-SEM imaging data that support the findings of this study are available from the corresponding author upon reasonable request.
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We thank S. Clerc-Rosset (EPFL) for processing the mouse brain tissue. We thank J. Kornfeld (MIT) for allowing the use of a flood-filling network that was trained on one of his SBEM datasets. We thank A. Eberle (Zeiss) for MultiSEM imaging our GCIB-SEM samples. We thank Y. Kubota (SOKENDAI) for providing the copper tape used in our ATUM collection tests. We thank W. Denk (Max Planck Institute) and M. Kormacheva (Max Planck Institute) for useful discussions. This work was funded by the Howard Hughes Medical Institute.
A patent on the GCIB-SEM technology has been filed by HHMI. M.J. is an employee of Google AI.
Peer review information Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–26 and Notes 1–3.
GCIB-SEM dataset of three 1-µm-thick sections of fly brain tissue; data were acquired with 6 × 6 × 4 nm voxels using InLens-SE detection.
GCIB-SEM dataset of three 500-nm-thick sections of mouse cortex tissue; data were acquired with 8 × 8 × 6 nm voxels using both InLens-SE (top) and ESB (bottom) detection.
GCIB-SEM dataset of ten 1-µm-thick sections of mouse cortex tissue; data were acquired with 8 × 8 × 6 nm voxels using ESB detection.
GCIB-SEM dataset of two 10-µm-thick hot-knife sections of mouse cortex tissue; data acquired with 10 × 10 × 12 nm voxels using both ESB (left) and InLens-SE (right) detection.
Flattening software and test dataset.
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Hayworth, K.J., Peale, D., Januszewski, M. et al. Gas cluster ion beam SEM for imaging of large tissue samples with 10 nm isotropic resolution. Nat Methods 17, 68–71 (2020). https://doi.org/10.1038/s41592-019-0641-2