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Imaging cellular ultrastructures using expansion microscopy (U-ExM)

Abstract

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

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The data that support the findings of this study are available from the corresponding authors upon request.

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Acknowledgements

We thank N. Klena for critical reading of the manuscript. We thank the BioImaging Center (University of Geneva) for help in image acquisition. We thank the Martinou lab and especially S. Zaganelli for helpful discussions and sharing of mitochondrial reagents. Human U2OS cells were a gift from E. Nigg (Biozentrum, University of Basel, Basel, Switzerland). D.G. and M.S.-C. are supported by the European Research Council (ERC; StG 715289 (ACCENT)). P.G., V.H., and M.L.G. are supported by the Swiss National Science Foundation (SNSF; PP00P3_157517). F.U.Z. and M.S. acknowledge support by the Deutsche Forschungsgemeinschaft (DFG) within the Collaborative Research Center 166 ReceptorLight (projects A04 and B04). M.U. is supported by the ERC (GA No. 692726 GlobalBioIm).

Author information

D.G., F.U.Z., M.S., V.H., and P.G. conceived and designed the project. M.S., V.H., and P.G. supervised the project. D.G. and F.U.Z. performed all ExM experiments. D.G. performed all U-ExM experiments with the help of S.B., as well as the data analysis. F.U.Z. performed the dSTORM imaging and the experiment and analysis involving clathrin-coated pits. J.H., J.-G.S., and M.R. performed and analyzed the STED imaging. D.F. and M.U. performed the 3D averaging. M.S.-C. initiated the U-ExM project. M.L.G. performed the plot profile of the polar transform showing the ninefold symmetry, as well as the r.m.s. calculation. E.S.B. helped in setting up ExM. All authors wrote and revised the final manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Markus Sauer or Virginie Hamel or Paul Guichard.

Supplementary information

  1. Supplementary text and figures

    Supplementary Figures 1–19

  2. Reporting Summary

  3. Supplementary Video 1

    U-ExM of isolated Chlamydomonas centrioles. Confocal (HyVolution) stack of immunolabeled Chlamydomonas centrioles in U-ExM. Magenta corresponds to α-tubulin, and green to PolyE. Scale bar, 1 µm.

  4. Supplementary Video 2

    3D rendering of isolated Chlamydomonas centrioles treated by U-ExM. 3D rendering of the confocal stack from Supplementary Video 1. Note the polyglutamylation signal (PolyE; green) along the microtubule triplets of the mature centrioles. Scale bar, 1 µm.

  5. Supplementary Video 3

    DyMIN imaging of U-ExM isolated Chlamydomonas centrioles. Stack of immunolabeled Chlamydomonas centrioles treated by U-ExM and imaged with DyMIN. Magenta corresponds to α-tubulin, and green to PolyE. Scale bar, 1 µm.

  6. Supplementary Video 4

    Supplementary Video 4U-ExM of Chlamydomonas cell. Confocal (HyVolution) stack of an immunolabeled Chlamydomonas cell treated by U-ExM. Magenta corresponds to α-tubulin, and green to PolyE.

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Further reading

Fig. 1: Centriole expansion with U-ExM.
Fig. 2: U-ExM reaches dSTORM precision limits.
Fig. 3: U-ExM applied to human cells.