Abstract
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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Data availability
The GCIB-SEM imaging data that support the findings of this study are available from the corresponding author upon reasonable request.
Code availability
GCIB-SEM flattening software and a small test dataset has been made available in the Supplementary Software and at Code Ocean (https://doi.org/10.24433/CO.4372524.v1)24.
Change history
04 December 2019
A Correction to this paper has been published: https://doi.org/10.1038/s41592-019-0695-1
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Acknowledgements
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.
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Authors and Affiliations
Contributions
K.J.H. performed experiments and wrote the manuscript. K.J.H., C.S.X. and H.F.H. conceived of the GCIB-SEM technique. K.J.H. and H.F.H. designed and built the prototype. K.J.H. and D.P. wrote the control software. K.J.H. wrote the analysis software. Z.L. provided fly brain tissue. G.W.K. provided mouse brain tissue. M.J. performed flood-fill network segmentations.
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Competing interests
A patent on the GCIB-SEM technology has been filed by HHMI. M.J. is an employee of Google AI.
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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.
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Supplementary information
Supplementary Information
Supplementary Figs. 1–26 and Notes 1–3.
Supplementary Video 1
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.
Supplementary Video 2
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.
Supplementary Video 3
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.
Supplementary Video 4
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.
Supplementary Software
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
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DOI: https://doi.org/10.1038/s41592-019-0641-2
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