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Three-dimensional cellular ultrastructure resolved by X-ray microscopy

Abstract

We developed an X-ray microscope using partially coherent object illumination instead of previously used quasi-incoherent illumination. The design permitted the incorporation of a cryogenic tilt stage, enabling tomography of frozen-hydrated, intact adherent cells. We obtained three-dimensional reconstructions of mouse adenocarcinoma cells at 36-nm (Rayleigh) and 70-nm (Fourier ring correlation) resolution, which allowed us to visualize the double nuclear membrane, nuclear pores, nuclear membrane channels, mitochondrial cristae and lysosomal inclusions.

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Figure 1: X-ray microscope designs.
Figure 2: X-ray images of a cell.
Figure 3: Volumetric rendering of cell cytoplasm.

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Acknowledgements

We thank B. Niemann for support and A. Leis for help with learning cryopreservation. This work was funded in part by the Human Frontier Science Program Research grant RGP0053/2005-C, the German Federal Ministry of Education and Research under contract 05KS4BY1/7, the intramural program of the US National Institutes of Health, including the National Institute of Arthritis and Musculoskeletal and Skin Diseases, and the National Cancer Institute, Center for Cancer Research, including Science Applications International Corporations Frederick contract HHSN26120080001E.

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Authors

Contributions

G.S. directed the X-ray microscopy project and with his colleagues designed and built the microscope. P.G. contributed to the design and construction of the microscope and collected all tomographic data. S.H. assisted with microscope construction, wrote the software to control the microscope and helped with some data collection. S.R. assisted with microscope construction and designed and constructed the zone plate objectives. F.M. performed most of the tomographic reconstructions. K.N. performed electron microscopy. J.B.H. adapted his software package Bsoft to permit reconstruction of the X-ray tomographic datasets and helped interpret the reconstructed images. W.G.M. prepared all of the specimens, collected the light microscopy data, helped collect the X-ray data and developed methods to grow the mouse cells on grids and preserve them by cryofixation. J.G.M. directed the cell biology project, helped design the study and wrote the paper.

Corresponding authors

Correspondence to Gerd Schneider or James G McNally.

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The authors declare no competing financial interests.

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Supplementary Figures 1–7 and Supplementary Protocol (PDF 2473 kb)

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Schneider, G., Guttmann, P., Heim, S. et al. Three-dimensional cellular ultrastructure resolved by X-ray microscopy. Nat Methods 7, 985–987 (2010). https://doi.org/10.1038/nmeth.1533

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