Article | Published:

Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues

Nature Biotechnology volume 34, pages 973981 (2016) | Download Citation

Abstract

The biology of multicellular organisms is coordinated across multiple size scales, from the subnanoscale of molecules to the macroscale, tissue-wide interconnectivity of cell populations. Here we introduce a method for super-resolution imaging of the multiscale organization of intact tissues. The method, called magnified analysis of the proteome (MAP), linearly expands entire organs fourfold while preserving their overall architecture and three-dimensional proteome organization. MAP is based on the observation that preventing crosslinking within and between endogenous proteins during hydrogel-tissue hybridization allows for natural expansion upon protein denaturation and dissociation. The expanded tissue preserves its protein content, its fine subcellular details, and its organ-scale intercellular connectivity. We use off-the-shelf antibodies for multiple rounds of immunolabeling and imaging of a tissue's magnified proteome, and our experiments demonstrate a success rate of 82% (100/122 antibodies tested). We show that specimen size can be reversibly modulated to image both inter-regional connections and fine synaptic architectures in the mouse brain.

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Acknowledgements

The authors thank the entire Chung laboratory for support and helpful discussions. K.C. was supported by Burroughs Wellcome Fund Career Awards at the Scientific Interface, the Searle Scholars Program, Packard award in Science and Engineering, JPB Foundation (PIIF and PNDRF) and NIH (1-U01-NS090473-01). Resources that may help enable general users to establish the methodology are freely available online (http://www.chunglabresources.org). K.C. is a co-founder of LifeCanvas Technologies, a startup that aims to help the research community adopt technologies developed by the Chung Laboratory.

Author information

Author notes

    • Taeyun Ku
    • , Justin Swaney
    •  & Jeong-Yoon Park

    These authors contributed equally to this work.

Affiliations

  1. Institute for Medical Engineering and Science, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.

    • Taeyun Ku
    • , Jeong-Yoon Park
    • , Alexandre Albanese
    • , Evan Murray
    • , Young-Gyun Park
    •  & Kwanghun Chung
  2. Picower Institute for Learning and Memory, MIT, Cambridge, Massachusetts, USA.

    • Taeyun Ku
    • , Jeong-Yoon Park
    • , Young-Gyun Park
    •  & Kwanghun Chung
  3. Department of Chemical Engineering, MIT, Cambridge, Massachusetts, USA.

    • Justin Swaney
    • , Jae Hun Cho
    •  & Kwanghun Chung
  4. Department of Neurosurgery, Gangnam Severance Hospital, Spine and Spinal Cord Institute, Yonsei University College of Medicine, Seoul, Republic of Korea.

    • Jeong-Yoon Park
  5. Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA.

    • Evan Murray
    •  & Kwanghun Chung
  6. Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA.

    • Vamsi Mangena
  7. Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA.

    • Jiapei Chen
  8. Broad Institute of Harvard University and MIT, Cambridge, Massachusetts, USA.

    • Kwanghun Chung

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Contributions

T.K., J.S., J.-Y.P., and K.C. designed the experiments and wrote the paper with input from other authors. T.K. stained and imaged mouse samples. J.S. performed the gel and cell experiments. T.K. and J.S. analyzed the data. J.-Y.P. prepared mouse tissues. J.-Y.P. and V.M. processed mouse MAP samples. A.A. performed the cell and organoid experiments. E.M., Y.-G.P., and T.K. performed the antibody validation test. J.H.C. performed stochastic electrotransport staining. Y.-G.P. and T.K. obtained synaptic images. J.-Y.P., V.M., T.K., and J.S. performed tracing. J.C. performed the gel experiment. K.C. supervised all aspects of the work.

Competing interests

K.C., T.K., J.S. and J.-Y.P. are coinventors on patent application owned by MIT covering the MAP technology (US Provisional Patent Application 62/330,018).

Corresponding author

Correspondence to Kwanghun Chung.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–3 and Supplementary Tables 1 and 2

Videos

  1. 1.

    Supplementary Video 1

    Exploration of fine cytoskeletal structures of a cortical neuron expressing NF-H.

  2. 2.

    Supplementary Video 2

    Visualization of SMI-312 fibers and TH-positive subcortical neuron.

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    Supplementary Video 3

    Examination of astrocyte-endothelial interactions and morphology using MAP.

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    Supplementary Video 4

    Visualization of NF-M fibers and spine-associated structures.

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    Supplementary Video 5

    Visualization of homer1 clusters in mouse cortex.

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    Supplementary Video 6

    Visualization of dense SMI-312 fiber bundles and fine TH structures.

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    Supplementary Video 7

    Tracing of a long-range TH fiber.

  8. 8.

    Supplementary Video 8

    Demonstration of SMI-312 fiber tracing.

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DOI

https://doi.org/10.1038/nbt.3641

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