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Expansion microscopy with conventional antibodies and fluorescent proteins

Nature Methods volume 13, pages 485–488 (2016)Cite this article

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Abstract

Expansion microscopy is a technique in which fluorophores on fixed specimens are linked to a swellable polymer that is physically expanded to enable super-resolution microscopy with ordinary microscopes. We have developed and characterized new methods for linking fluorophores to the polymer that now enable expansion microscopy with conventional fluorescently labeled antibodies and fluorescent proteins. Our methods simplify the procedure and expand the palette of compatible labels, allowing rapid dissemination of the technique.

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Figure 1: Schematic illustration of expansion microscopy and label retention strategies.
Figure 2: Confocal fluorescence images of expanded cultured cells.
Figure 3: Images of expanded mouse brain tissue prepared using the MA-NHS treatment method.

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Acknowledgements

This work is supported by the University of Washington (J.C.V.), a Burroughs-Wellcome Career Award at the Scientific Interface (J.C.V.), an NSF Graduate Research Fellowship DGE-1256082 (T.J.C.), and by NIH grants EY10699 and EY17101 (R.O.L.W.). The authors would like to thank L. Wordeman (University of Washington, Seattle, WA) for providing the PtK1 cell line and anti-HEC1 antibody, for access to an electroporator, and for helpful discussions; K. Oda (University of Washington, Seattle, WA) for performing the cardiac perfusion of mice; T. Rapoport (Harvard Medical School, Boston, MA) for the Sec61β-GFP plasmid; E. Boyden, F. Chen, and P. Tillberg (MIT, Boston, MA) for conducting an ExM workshop; and the Biology Imaging Facility at the University of Washington for imaging assistance.

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Author notes

  1. Tyler J Chozinski and Aaron R Halpern: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, University of Washington, Seattle, Washington, USA

    Tyler J Chozinski, Aaron R Halpern, Hyeon-Jin Kim, Grant J Tremel & Joshua C Vaughan

  2. Department of Biological Structure, University of Washington, Seattle, Washington, USA

    Haruhisa Okawa & Rachel O L Wong

  3. Department of Physiology and Biophysics, University of Washington, Seattle, Washington, USA

    Joshua C Vaughan

Authors
  1. Tyler J Chozinski
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  2. Aaron R Halpern
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  3. Haruhisa Okawa
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  4. Hyeon-Jin Kim
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  5. Grant J Tremel
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  6. Rachel O L Wong
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  7. Joshua C Vaughan
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Contributions

T.J.C., A.R.H., H.O., R.O.L.W., and J.C.V. designed the experiments. T.J.C., A.R.H., H.O., H.-J.K., and G.J.T. performed the experiments and analysis. T.J.C., A.R.H., and J.C.V. wrote the paper and all authors commented on the manuscript. J.C.V. supervised the project.

Corresponding author

Correspondence to Joshua C Vaughan.

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Competing interests

The authors have filed a patent application (U.S. Provisional 62/311, 638).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–21, Supplementary Table 1 and Supplementary Protocol (PDF 3472 kb)

Supplementary Analysis

Example data and processing scripts for pre-expansion/post-expansion distortion analysis. (ZIP 4233 kb)

3D animation of expanded brain slice shown in Fig. 3 a-f.

The animation begins as 29 × 13 μm and then zooms to show a view of 4.6 × 2.7 μm (all distances are in pre-expansion dimensions). (MOV 14725 kb)

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Chozinski, T., Halpern, A., Okawa, H. et al. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat Methods 13, 485–488 (2016). https://doi.org/10.1038/nmeth.3833

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