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

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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Figure 1: Magnified and accessible 3D proteome library of whole intact organs.
Figure 2: Comparison of multiscale architectures before and after MAP processing.
Figure 3: Super-resolution imaging showing the 3D proteome library and subcellular details of MAP-processed intact tissue.
Figure 4: Multiplexed staining of MAP-processed tissue.
Figure 5: Intercellular connectivity and its reconstruction at single-fiber resolution in MAP-processed tissue.
Figure 6: Immunolabeling and imaging of thick MAP-processed tissues.

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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

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Authors

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.

Corresponding author

Correspondence to Kwanghun Chung.

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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).

Integrated supplementary information

Supplementary Figure 1 Expansion of various organs with MAP.

Photographs of intact organs being MAP processed. Organs were harvested after perfusion using PBS solution containing 4% PFA, 30% AA, 0.05% BA, 5% SA, and 0.1% VA-044. After allowing 2 days for chemical diffusion at 4°C, hydrogel-tissue hybridization was performed at 50°C for 2 h. Hydrogel-embedded organs were incubated in a 200 mM SDS and 50 mM sodium sulfite PBS solution for at least 24 h at 70°C and 12 h at 95°C. Denatured tissues were incubated in 100 ml DI water at room temperature for at least 36 h with gentle shaking. Scale bars, 10 mm.

Supplementary Figure 2 Validation of commercial antibodies targeting cell-type markers in MAP-processed tissues.

Fluorescence images from various commercial antibodies targeting cell-type markers tested in both MAP and control samples. Control and MAP samples were sectioned to 100-μm thickness and then stained after denaturation. Primary incubation was performed for 12 h at 37°C with gentle shaking followed by a two-step wash of 2 h each in PBST. Secondary incubation was performed for 6 h at 37°C, followed by a 2-h wash in PBST, 30 min in 1:50,000 DAPI solution, then another 2-h wash in PBST. To determine specificity, antibodies targeting the same antigen were tested simultaneously in a single tissue using separate color channels when possible. Images were acquired with our Olympus confocal microscope with the following settings: 550 HV, 10 μs pixel−1 dwell time, 1,024 × 1,024 resolution, 0% offset, and laser power sufficient to nearly saturate signals. A 20×, 0.95 NA water-immersion objective was used. MP, Millipore; BL, BioLegend, CST, Cell Signaling Technology. Scale bars, 20 μm.

Supplementary Figure 3 Validation of commercial antibodies targeting neurofilament markers in MAP-processed tissues.

Fluorescence images from various commercial antibodies targeting neurofilament markers tested in both MAP and control samples. Images were obtained using the same method as Supplementary Figure 2. Scale bars, 20 μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Tables 1 and 2 (PDF 4552 kb)

Supplementary Video 1

Exploration of fine cytoskeletal structures of a cortical neuron expressing NF-H. (MOV 13046 kb)

Supplementary Video 2

Visualization of SMI-312 fibers and TH-positive subcortical neuron. (MOV 14733 kb)

Supplementary Video 3

Examination of astrocyte-endothelial interactions and morphology using MAP. (MOV 6265 kb)

Supplementary Video 4

Visualization of NF-M fibers and spine-associated structures. (MOV 23717 kb)

Supplementary Video 5

Visualization of homer1 clusters in mouse cortex. (MOV 22712 kb)

Supplementary Video 6

Visualization of dense SMI-312 fiber bundles and fine TH structures. (MOV 22499 kb)

Supplementary Video 7

Tracing of a long-range TH fiber. (MOV 20280 kb)

Supplementary Video 8

Demonstration of SMI-312 fiber tracing. (MOV 22201 kb)

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Ku, T., Swaney, J., Park, JY. et al. Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues. Nat Biotechnol 34, 973–981 (2016). https://doi.org/10.1038/nbt.3641

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