Abstract

We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel–specimen composite by 4.5× in linear dimension. Here we describe iterative expansion microscopy (iExM), in which a sample is expanded 20×. After preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by the first expansion, and the sample is expanded again. iExM expands biological specimens 4.5 × 4.5, or 20×, and enables 25-nm-resolution imaging of cells and tissues on conventional microscopes. We used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in mouse brain circuitry.

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Acknowledgements

E.S.B. was funded by the HHMI-Simons Faculty Scholars Program; the NIH Director′s Pioneer Award 1DP1NS087724; the New York Stem Cell Foundation-Robertson Award; the US Army Research Laboratory and the US Army Research Office under contract/grant number W911NF1510548; US-Israel Binational Science Foundation Grant 2014509; the Picower Institute Innovation Fund; IARPA D16PC00008; NIH grants 1R01MH110932, 1R43MH101943, 1R01MH103910, 1R01EY023173, and 2R01DA029639; the IET A.F. Harvey Prize; the Open Philanthropy Project; the Halis Family Foundation; and the MIT Media Lab. J.-B.C. was supported by the Simons Postdoctoral Fellowship. F.C. was supported by the NSF Fellowship and Poitras Fellowship. Y.-G.Y., J.S.K., and H.-J.S. were supported by Samsung Scholarships. P.W.T. and A.T.W. were supported by Hertz Foundation fellowships. Confocal imaging was performed in the W.M. Keck Facility for Biological Imaging at the Whitehead Institute for Biomedical Research J.-B.C. was supported by the Center for Neuroscience Imaging Research. D.C. was funded by NIH grants R21GM114852 and R01MH110932. We acknowledge W. Salmon (MIT) for her assistance with confocal imaging. STORM imaging shown in Figure 2o was performed in the Center for Brain Science at Harvard University. We acknowledge E. Garner, C. Wivagg, and S. Turney (Harvard) for allowing us to use the N-STORM microscope and their assistance with STORM imaging. We acknowledge D. Park (MIT) for assistance with the preparation of cultured neurons. We also acknowledge S. Shim, Y. Sigal, C. Speer, M. Thanawala, D. Kim, M. Sauer, and S. Alon for helpful discussions. We acknowledge University of Michigan, Ann Arbor for providing antibodies against Brainbow fluorescent proteins.

Author information

Affiliations

  1. Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.

    • Jae-Byum Chang
    • , Young-Gyu Yoon
    • , Erica E Jung
    • , Shoh Asano
    •  & Edward S Boyden
  2. Department of Biomedical Engineering, Sungkyunkwan University, Suwon, Korea.

    • Jae-Byum Chang
  3. Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA.

    • Fei Chen
    • , Asmamaw T Wassie
    •  & Edward S Boyden
  4. Department of Electrical Engineering and Computer Science, MIT, Cambridge, Massachusetts, USA.

    • Young-Gyu Yoon
    •  & Paul W Tillberg
  5. Harvard Center for Advanced Imaging, Harvard University, Cambridge, Massachusetts, USA.

    • Hazen Babcock
  6. John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.

    • Jeong Seuk Kang
  7. Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA.

    • Ho-Jun Suk
  8. Department of Mechanical Engineering, MIT, Cambridge, Massachusetts, USA.

    • Nikita Pak
  9. Department of Cell and Developmental Biology, Medical School, Biophysics Research Division, LS&A, University of Michigan, Ann Arbor, Michigan, USA.

    • Dawen Cai
  10. McGovern Institute, MIT, Cambridge, Massachusetts, USA.

    • Edward S Boyden
  11. Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA.

    • Edward S Boyden

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Contributions

E.S.B. and J.-B.C. conceived the main idea and designed experiments. P.W.T. conceived hp-iExM strategy. J.-B.C. performed immunostaining and expanded specimens. J.-B.C., F.C., and A.W. developed re-embedding process. J.-B.C. calculated RMS error of iExM. F.C. conceived signal amplification methods. E.E.J. performed immunostaining. Y.-G.Y. performed deconvolution and denoising and developed the iExM simulator. H.-J.S. and N.P. performed the brainbow virus injection and perfusion. Y.-G.Y. and S.A. created 3D videos. H.B. contributed STORM data in Figure 2a–c. J.-B.C. performed STORM imaging for Figure 2o,p. D.C. provided antibodies against Brainbow fluorescent proteins. J.-B.C. and J.S.K. imaged samples and performed image processing. J.-B.C. performed statistical analysis. E.S.B. and J.-B.C. wrote the paper. All authors contributed to editing of the paper. E.S.B. supervised this work.

Competing interests

E.S.B., J.-B.C., F.C., and P.W.T. have applied for a patent on iExM (US application 20160305856 A1). E.S.B. is cofounder of Expansion Technologies, a company that aims to provide expansion microscopy kits and services to the community.

Corresponding author

Correspondence to Edward S Boyden.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15, Supplementary Tables 1–14, and Supplementary Notes 1–8

  2. 2.

    Supplementary Protocol

    Supplementary Protocol

Zip files

  1. 1.

    Supplementary Software

    iExM simulator. MATLAB program simulating iExM of a microtubule labeled with a primary antibody and DNA-conjugated secondary antibody.

Videos

  1. 1.

    3-D visualization of microtubules:

    3-D visualization of the z-stack confocal image of beta tubulin-stained BS-C-1 cells after 20-fold expansion via iExM shown in Fig. 2g.

  2. 2.

    Zoomed-in 3-D video of the center region of Supple. Video 1

    3-D visualization of the center region of the z-stack shown in Supplementary Video 1 and Fig. 2g. Red numbers of the bounding box of the video show scales in micron.

  3. 3.

    3-D visualization of Homer1 and Bassoon in medial pallidum

    3-D visualization of the z-stack confocal image of Homer1(green)/Bassoon(red) stained medial pallidum after 16-fold expansion via iExM shown in Fig. 3l-o. Red numbers of the bounding box of the video show scales in micron.

  4. 4.

    3-D visualization of GABAAR and Bassoon in globus pallidus

    3-D visualization of the z-stack confocal image of GABAARα1α2/Bassoon stained globus pallidus after 16-fold expansion via iExM shown in Supplementary Fig. 10a.

  5. 5.

    3-D visualization and surface rendering of Brainbow mouse hippocampus

    Z-stack confocal image of the molecular layer of the mouse hippocampal dentate gyrus after immunostaining of EYFP (blue) and mCherry (green) and 20-fold expansion via iExM shown in Fig. 4c. This video also shows 3-D visualization and surface rendering of the stack.

  6. 6.

    3-D visualization of Brainbow mouse hippocampus

    3-D visualization of the z-stack confocal image of the molecular layer of the mouse hippocampal dentate gyrus after immunostaining of mCherry, TagBFP, and mTFP and 16-fold expansion via iExM shown in Supplementary Fig. 12h.

  7. 7.

    3-D visualization of Brainbow mouse hippocampus showing a branching dendrite

    3-D visualization of the z-stack confocal image of the molecular layer of the mouse hippocampal dentate gyrus after immunostaining of mCherry, TagBFP, mTFP, and EYFP and 20-fold expansion via iExM shown in Fig. 4g.

  8. 8.

    Another 3-D visualization of Brainbow mouse hippocampus showing a branching dendrite

    3-D visualization of the z-stack confocal image of the molecular layer of the mouse hippocampal dentate gyrus after immunostaining of mCherry, TagBFP, and mTFP and 16-fold expansion via iExM shown in Supplementary Fig. 13h.

  9. 9.

    Another 3-D visualization of Brainbow mouse hippocampus

    3-D visualization of the z-stack confocal image of the molecular layer of the mouse hippocampal dentate gyrus after immunostaining of mCherry, TagBFP, and mTFP and 16-fold expansion via iExM shown in Supplementary Fig. 13p.

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https://doi.org/10.1038/nmeth.4261

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