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Protection of tissue physicochemical properties using polyfunctional crosslinkers


Understanding complex biological systems requires the system-wide characterization of both molecular and cellular features. Existing methods for spatial mapping of biomolecules in intact tissues suffer from information loss caused by degradation and tissue damage. We report a tissue transformation strategy named stabilization under harsh conditions via intramolecular epoxide linkages to prevent degradation (SHIELD), which uses a flexible polyepoxide to form controlled intra- and intermolecular cross-link with biomolecules. SHIELD preserves protein fluorescence and antigenicity, transcripts and tissue architecture under a wide range of harsh conditions. We applied SHIELD to interrogate system-level wiring, synaptic architecture, and molecular features of virally labeled neurons and their targets in mouse at single-cell resolution. We also demonstrated rapid three-dimensional phenotyping of core needle biopsies and human brain cells. SHIELD enables rapid, multiscale, integrated molecular phenotyping of both animal and clinical tissues.

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Figure 1: Protection of GFP activity with polyepoxide cross-linkers.
Figure 2: SHIELD preserves fluorescent protein signals, proteins, transcripts and their probe-binding affinities.
Figure 3: SHIELD protects tissue architecture against physical and chemical stressors.
Figure 4: SHIELD enables 3D imaging of various structures and cell types and their morphological details in human brain tissue.
Figure 5: SHIELD enables new tissue phenotyping approaches.
Figure 6: SHIELD enables integrated circuit reconstruction at single cell resolution.

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The authors thank the entire Chung laboratory for support and discussions. We acknowledge S. Speck, A. Tran, J. Senecal, W. Guan and L. Kamentsky for their contribution to image processing and data analysis. K.C. was supported by the Burroughs Wellcome Fund Career Awards at the Scientific Interface, Searle Scholars Program, Packard Award in Science and Engineering, NARSAD Young Investigator Award, McKnight Foundation Technology Award, JPB Foundation (PIIF and PNDRF), NCSOFT Cultural Foundation and NIH (1-DP2-ES027992). R.C. was supported by a SCSB fellowship. B.K.L. was supported by the Klingenstein Foundation, Searle Scholar program (Kinship Foundation), Whitehall Foundation, NARSAD Young Investigator Award and grants from NIMH (R01MH107742, R01MH108594, U01MH114829). V.L. is supported by Anandamahidol Foundation fellowship. M.P.F. was partially supported by NIA P50 AG005134. H.J.K. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund, which supported the work. H.W.Q. was supported in part by a Department of Energy Computational Science Graduate Fellowship (DOE-CSGF). This work was carried out in part using computational resources from the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant ACI-1548562. This work used the XStream computational resource, supported by the National Science Foundation Major Research Instrumentation program (ACI-1429830). S.-C.C. and J.W. were supported by HKSAR Research Grants Council (RGC) General Research Fund (GRF), number 14201214. K.C. is a cofounder of LifeCanvas Technologies, a startup that aims to help the research community adopt technologies developed by the Chung Laboratory.

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Authors and Affiliations



K.C. conceived the idea. Y.-G.P., C.H.S., R.C., D.H.Y., and K.C. designed the experiments and wrote the paper with input from other authors. Y.-G.P. led development of SHIELD-SWITCH and SHIELD-MAP methods. H.J.K and H.W.Q. performed the molecular simulation in Figure 1. R.C. performed the physicochemical analysis in Figure 1 and quantified the fluorescence preservation in Figure 2. R.C. and M.M. performed fluorescent protein experiments in Figure 2. M.M. and R.C. acquired and analyzed the virus-labeled neuronal images in Figure 2. C.H.S. and Y.-G.P. performed the immunoreactivity experiments and autofluorescence quantification in Figure 2. R.C. performed the multi-round immunostaining in Figure 2. C.H.S. and R.C. designed and performed the FISH experiments and protein analyses. Y.-G.P. and C.H.S. characterized physical properties of SHIELD tissue in Figure 3. C.H.S. and G.D. designed SHIELD processing for postmortem human brain tissues and performed immunostaining in Figure 4. H.C. built temporally focused line-scanning microscope and acquired the large human brain slab image in Figure 4. D.H.Y. and Y.-G.P. established the pipeline for 3D phenotyping of the biopsy samples in Figure 5. T.K. performed the isotropic expansion analysis in Figure 5 and contributed to the development of SHIELD-MAP method. N.B.E. conducted light-sheet microscope imaging in Figure 6. Y.-G.P. and C.H.S. performed neuronal fiber tracing, and Y.-G.P performed imaging and analysis in Figure 6. H.C.O. and W.T. helped sample preparation. M.C.T. and H.L.P. provided purified GFP. X.J. and T.R.G. provided tumor tissues. S.C. and J.W. developed the oscillating blade microtome. M.P.F provided human brain tissues and validated the human brain data. V.L. and B.K.L. provided the virus, the virus labeled tissues, and helpful discussion. K.C. supervised all aspects of the work.

Corresponding author

Correspondence to Kwanghun Chung.

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Competing interests

K.C. is a co-inventor on patent application owned by MIT covering the SHIELD and SWITCH technology (PCT/US2016/064538).

Integrated supplementary information

Supplementary Figure 1 Mass spectrometry characterization of epoxide reactions.

(a) Degree of amine reactivity of bovine serum albumin (BSA) with epoxide molecules having different numbers of epoxide groups: 1 (GME), 2 (EGDGE, 1,4-BDE, DGDE), 3 (TGE), 4 (PEGE), 5 (P3PE) and with paraformaldehyde (PFA) and glutaraldehyde (GA). N=3 independent experiments. Mean +/- standard error mean was used for this bar graph. (b-c) MALDI-TOF spectra of BSA (b) or GFP (c) reacted with GME and P3PE. Corresponding mass shifts indicate a total of ~92 GME or ~45 P3PE molecules crosslinked with BSA, implying that single covalently attached P3PE molecules bridges ~2 epoxide-reactive residues (b). A similar calculation shows that roughly 1.5 available epoxide-reactive GFP side-chains are bridged by P3PE (c). Mass peak values are indicated as numbers in kDa. (d, e) DNA oligos reacted with epoxides at the reaction conditions used for tissue processing (pH 10, 0.1 M sodium carbonate buffer). MALDI-TOF spectra of (dA)15 (d) and (dC)15 oligos (e) show mass shifts associated respectively with multiple GME or P3PE epoxide crosslinks, respectively. Peak broadening was observed for P3PE crosslinked oligos indicating the formation of abundant salt adducts with polydisperse P3PE.

Supplementary Figure 2 Preservation of endogenous proteins and their probe-binding affinities in SHIELD tissue.

(a) Venn diagram of antigenicity test results. All 53 tested antibodies compatible with PFA-fixed control tissue worked in SHIELD tissue. (b) Protein loss assay in the high-temperature tissue clearing condition (200 mM SDS, 70°C, 12 hr). N = 3 tissues. (c, d) FP preservation in SHIELD-processed tissues after multiple rounds of staining and destaining. (c) Endogenous YFP signal retention of PFA and SHIELD tissues after destaining treatments corresponding to 5 rounds of stainings. N=4 tissues. (d) Multiround staining images from the hippocampal region. Break lines indicate destaining steps. For the second round, secondary antibodies were added to confirm complete unbinding of the primary antibodies imaged at the first round. Thy1-H+ YFP mouse SHIELD tissue was used. Scale bars = 100 μm (e) Representative 21 immunofluorescence images in PFA, GA, and SHIELD-processed tissues. Scale bar = 100 μm. The same imaging and display settings were used for each antibody. (f) The contrast-adjusted reproduction of Figure 2i. The selected images were adjusted for better visual comparison. Scale bar = 20 μm (g) Representative images comparing the immunofluorescence of MBP and NF-H in uncleared PFA, GA, and SHIELD tissues. To exclude the effect of tissue clearing on antigenicity, uncleared sections were used. Scale bar = 20 μm. MBP, myelin basic protein; NF-H, neurofilament-H. (h) SHIELD maintained a SNR of MBP and NF-H immunofluorescences similar to that of the PFA-control in immunohistochemistry, indicating minimal epitope damage by P3PE crosslinking. N = 4 tissues. Unpaired T-test, *P < 0.05. Mean +/- standard error mean was used for all the bar graphs.

Supplementary Figure 3 Detection of transcripts in SHIELD tissue by fluorescence in situ hybridization–hybridization chain reaction (FISH-HCR) with various probe designs.

(a-c) FISH-HCR with 18 nt (a-b) and 50 nt (c) probes for YFP mRNA. The endogenous YFP fluorescence signal was well co-localized with FISH Cy5 signals in individual cells. (d-h) FISH-HCR on mRNA transcripts of cell-type marker proteins and their corresponding ISH images from the Allen brain atlas (© 2015 Allen Institute for Brain Science. Allen Brain Atlas API. Available from: (d, e) FISH-HCR for glutamate decarboxylase (GAD1, an inhibitory neuronal marker) using 22nt (d) and 35nt (e) probes. (f) Somatostatin (SST), (g) neuropeptide Y (NPY), and (h) vesicular glutamate transporter (vGluT2, an excitatory neuronal marker) mRNAs were successfully detected by FISH-HCR in cleared SHIELD-tissue. Scale bar = 1 mm. (i, j) GFP signal was preserved better in SHIELD tissue than in tissues processed with EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) after clearing. (i) Bar graph. Unpaired T-test, **P<0.01. N=3 tissues. (j) Representative images. Scale bar = 1 mm. Mean +/- standard error mean is used for this graph. Data in panel j was repeated 3 times independently to make a bar graph in panel i.

Supplementary Figure 4 Structural integrity of SHIELD tissue.

(a-b) Autofluorescence (a) and YFP (b) images of 1mm-thick tissues from different anatomical coordinates of mouse brain hemisphere before and after clearing, and after additional 70°C destaining step (300mM SDS, 2 hr). Images that were not included in Figure 3a are presented here. The contour of the uncleared sections are marked with yellow dotted lines in subsequent images. In YFP images, numbers on the top right corners indicate the intensity gain from image display range. PACT images after 70°C destaining step are not included because the tissues melted. Scale bars = 1 mm.

Supplementary Figure 5 Whole organ processing and clearing with SHIELD.

(a) SWITCH chemistry in SHIELD processing. Epoxy-amine reactivity at 37°C pH 10 condition (SWITCH-ON) was 10-fold higher than reactivity at 4°C pH 7.4 condition (SWITCH-OFF). Unpaired T-test, *P < 0.05, N = 3 independent experiments. Mean +/- standard error mean is used for this bar graph. (b) Schematic diagram of whole organ SHIELD processing using SWITCH chemistry. The initial incubation of tissue in the SWITCH-OFF condition ensures complete and homogeneous distribution of epoxide molecules across the tissue volume. Subsequent SWITCH-ON condition initiates synchronized crosslinking throughout the tissue volume. (c) Comparison of SHIELD tissue processed with (right) or without SWITCH (left). Note that the control sample shows opaque tissue layer at its surface even after clearing, suggesting overfixation at the tissue surface. SHIELD tissue fixed with SWITCH chemistry is uniformly transparent throughout its volume after clearing. Grid = 1 mm. (d) SHIELD-processed mouse organs before (left panels) and after clearing (right panels). Grid = 1 mm. (e) 3D visualization of an intestine of ChAT-EGFP transgenic mouse showing processes of ChAT+ motor neurons innervating the intestine. Scale bars = 1 mm (left, lower right), 50 μm (upper right).

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Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 1678 kb)

Life Sciences Reporting Summary (PDF 163 kb)

Supplementary Tables and Note

Supplementary Tables 1–3 and Supplementary Note (PDF 538 kb)

Supplementary Video 1

A series of xy-plane images showing uniform (dT)50-Cy3 FISH signal in a SHIELD-processed block. (MP4 19074 kb)

Supplementary Video 2

3D autofluorescence (white) and YFP (green) signals from Thy1-H line mouse hemispheres processed with PACT, iDISCO+ and SHIELD. (MP4 22203 kb)

Supplementary Video 3

3D rendering of a SHIELD-processed human brain slab (size: 22 mm × 38 mm × 1.3 mm) highlighting nuclei (green) and blood vessels (red). (MP4 173891 kb)

Supplementary Video 4

3D rendering of GFAP (green) and CR (red) immunolabeling of a 1-mm-thick SHIELD-processed human cortical coronal block. (MP4 104641 kb)

Supplementary Video 5

3D rendering showing a fresh biopsy sample processed with the SHIELD-based rapid (<4 h) phenotyping pipeline. (MP4 13714 kb)

Supplementary Video 6

Mouse kidney tumor tissue processed with a SHIELD-based rapid phenotyping pipeline, showing anti-Ki-67 immunostaining (green) and lectin labeling (red). (MP4 29707 kb)

Supplementary Video 7

A series of optical sections of SHIELD-MAP tissue from the surface to a depth of 5 mm. (MP4 122726 kb)

Supplementary Video 8

3D rendering of a reconstructed YFP-expressing neuron in a Thy1-YFP H line mouse brain cortex using SHIELD-MAP. (MP4 130548 kb)

Supplementary Video 9

3D rendering of hemisphere showing labeled GPe-PV+ circuitry. (MP4 203075 kb)

Supplementary Video 10

3D rendering of 1-mm-thick mouse tissue showing labeled GPe-PV+ circuitry after first-round GAD1-FISH (cyan) staining. (MP4 112693 kb)

Supplementary Video 11

3D super-resolution images of putative axosomatic connectivity. (MP4 71545 kb)

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Park, YG., Sohn, C., Chen, R. et al. Protection of tissue physicochemical properties using polyfunctional crosslinkers. Nat Biotechnol 37, 73–83 (2019).

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