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.
Subscribe to Journal
Get full journal access for 1 year
only $20.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Protein Data Bank
Masland, R.H. Neuronal cell types. Curr. Biol. 14, R497–R500 (2004).
Kepecs, A. & Fishell, G. Interneuron cell types are fit to function. Nature 505, 318–326 (2014).
Alivisatos, A.P. et al. The brain activity map. Science 339, 1284–1285 (2013).
DeFelipe, J. From the connectome to the synaptome: an epic love story. Science 330, 1198–1201 (2010).
Zingg, B. et al. Neural networks of the mouse neocortex. Cell 156, 1096–1111 (2014).
Kasthuri, N. et al. Saturated reconstruction of a volume of neocortex. Cell 162, 648–661 (2015).
Crosetto, N., Bienko, M. & van Oudenaarden, A. Spatially resolved transcriptomics and beyond. Nat. Rev. Genet. 16, 57–66 (2015).
Shah, S., Lubeck, E., Zhou, W. & Cai, L. In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus. Neuron 92, 342–357 (2016).
Giesen, C. et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat. Methods 11, 417–422 (2014).
Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332–337 (2013).
Susaki, E.A. & Ueda, H.R. Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals. Cell Chem. Biol. 23, 137–157 (2016).
Sylwestrak, E.L., Rajasethupathy, P., Wright, M.A., Jaffe, A. & Deisseroth, K. Multiplexed intact-tissue transcriptional analysis at cellular resolution. Cell 164, 792–804 (2016).
Ku, T. et al. Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues. Nat. Biotechnol. 34, 973–981 (2016).
Murray, E. et al. Simple, scalable proteomic imaging for high-dimensional profiling of intact systems. Cell 163, 1500–1514 (2015).
Renier, N. et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 (2014).
Pan, C. et al. Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat. Methods 13, 859–867 (2016).
Tainaka, K., Kuno, A., Kubota, S.I., Murakami, T. & Ueda, H.R. Chemical principles in tissue clearing and staining protocols for whole-body cell profiling. Annu. Rev. Cell Dev. Biol. 32, 713–741 (2016).
Ertürk, A. et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat. Protoc. 7, 1983–1995 (2012).
Chen, F., Tillberg, P.W. & Boyden, E.S. Expansion microscopy. Science 347, 543–548 (2015).
Vincek, V., Nassiri, M., Nadji, M. & Morales, A.R. A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples. Lab. Invest. 83, 1427–1435 (2003).
Pelegri-O'Day, E.M., Lin, E.W. & Maynard, H.D. Therapeutic protein-polymer conjugates: advancing beyond PEGylation. J. Am. Chem. Soc. 136, 14323–14332 (2014).
Kabanov, A.V. & Vinogradov, S.V. Nanogels as pharmaceutical carriers: finite networks of infinite capabilities. Angew. Chem. Int. Ed. Engl. 48, 5418–5429 (2009).
Sheldon, R.A. & van Pelt, S. Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soc. Rev. 42, 6223–6235 (2013).
Hartmann, M. Ordered mesoporous materials for bioadsorption and biocatalysis. Chem. Mater. 17, 4577–4593 (2005).
Mateo, C. et al. Some special features of glyoxyl supports to immobilize proteins. Enzyme Microb. Technol. 37, 456–462 (2005).
Migneault, I., Dartiguenave, C., Bertrand, M.J. & Waldron, K.C. Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. Biotechniques 37, 790–796, 798–802 (2004).
Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).
Kim, S.-Y. et al. Stochastic electrotransport selectively enhances the transport of highly electromobile molecules. Proc. Natl. Acad. Sci. USA 112, E6274–E6283 (2015).
Tsien, R.Y. Fluorescent probes of cell signaling. Annu. Rev. Neurosci. 12, 227–253 (1989).
Back, J.F., Oakenfull, D. & Smith, M.B. Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry 18, 5191–5196 (1979).
Mason, J.T. & O'Leary, T.J. Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation. J. Histochem. Cytochem. 39, 225–229 (1991).
Enoki, S., Saeki, K., Maki, K. & Kuwajima, K. Acid denaturation and refolding of green fluorescent protein. Biochemistry 43, 14238–14248 (2004).
Fowler, C.B., Evers, D.L., O'Leary, T.J. & Mason, J.T. Antigen retrieval causes protein unfolding: evidence for a linear epitope model of recovered immunoreactivity. J. Histochem. Cytochem. 59, 366–381 (2011).
Hama, H. et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci. 14, 1481–1488 (2011).
Hopwood, D. Theoretical and practical aspects of glutaraldehyde fixation. Histochem. J. 4, 267–303 (1972).
Lee, K., Choi, S., Yang, C., Wu, H.-C. & Yu, J. Autofluorescence generation and elimination: a lesson from glutaraldehyde. Chem. Commun. (Camb.) 49, 3028–3030 (2013).
Deforce, D.L.D., Ryniers, F.P.K., van den Eeckhout, E.G., Lemière, F. & Esmans, E.L. Analysis of DNA adducts in DNA hydrolysates by capillary zone electrophoresis and capillary zone electrophoresis-electrospray mass spectrometry. Anal. Chem. 68, 3575–3584 (1996).
Pena, J.T.G. et al. miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat. Methods 6, 139–141 (2009).
Pappa, V.I. et al. Role of image-guided core-needle biopsy in the management of patients with lymphoma. J. Clin. Oncol. 14, 2427–2430 (1996).
Nojima, S. et al. CUBIC pathology: three-dimensional imaging for pathological diagnosis. Sci. Rep. 7, 9269 (2017).
Tanaka, N. et al. Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity. Nat. Biomed. Eng. 1, 796–806 (2017).
Glaser, A.K. et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat. Biomed. Eng. 1, 1–10 (2017).
Tillberg, P.W. et al. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat. Biotechnol. 34, 987–992 (2016).
Dumitriu, D., Rodriguez, A. & Morrison, J.H. High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy. Nat. Protoc. 6, 1391–1411 (2011).
Ke, M.-T. Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent. Cell Rep. 14, 2718–2732 (2016).
Gerfen, C.R. & Wilson, C.J. in Handbook of Chemical Neuroanatomy 12, 371–468 (Elsevier, 1996).
Obeso, J.A., Rodriguez-Oroz, M.C., Stamelou, M., Bhatia, K.P. & Burn, D.J. The expanding universe of disorders of the basal ganglia. Lancet 384, 523–531 (2014).
Saunders, A., Huang, K.W. & Sabatini, B.L. Globus pallidus externus neurons expressing parvalbumin interconnect the subthalamic nucleus and striatal interneurons. PLoS One 11, e0149798 (2016).
Knowland, D. et al. Distinct ventral pallidal neural populations mediate separate symptoms of depression. Cell 170, 284–297.e18 (2017).
Ye, L. et al. Wiring and molecular features of prefrontal ensembles representing distinct experiences. Cell 165, 1776–1788 (2016).
Economo, M.N. et al. A platform for brain-wide imaging and reconstruction of individual neurons. Elife 5, e10566 (2016).
Luo, L., Callaway, E.M. & Svoboda, K. Genetic dissection of neural circuits: a decade of progress. Neuron 98, 256–281 (2018).
Chen, K.H., Boettiger, A.N., Moffitt, J.R., Wang, S. & Zhuang, X. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).
Beliveau, B.J. et al. Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat. Commun. 6, 7147 (2015).
Spurr, A.R. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31–43 (1969).
Halgren, T.A. Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94. J. Comput. Chem. 17, 490–519 (1996).
Halgren, T.A. Merck molecular force field. II. MMFF94 van der Waals and electrostatic parameters for intermolecular interactions. J. Comput. Chem. 17, 520–552 (1996).
Arpino, J.A.J., Rizkallah, P.J. & Jones, D.D. Crystal structure of enhanced green fluorescent protein to 1.35 Å resolution reveals alternative conformations for Glu222. PLoS One 7, e47132 (2012).
Ryckaert, J.-P., Ciccotti, G. & Berendsen, H.J.C. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J. Comput. Phys. 23, 327–341 (1977).
Yang, B. et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158, 945–958 (2014).
Renier, N. et al. Mapping of brain activity by automated volume analysis of immediate early genes. Cell 165, 1789–1802 (2016).
Choi, H.M.T., Beck, V.A. & Pierce, N.A. Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability. ACS Nano 8, 4284–4294 (2014).
Chen, F. et al. Nanoscale imaging of RNA with expansion microscopy. Nat. Methods 13, 679–684 (2016).
Shah, S. et al. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing. Development 143, 2862–2867 (2016).
Moffitt, J.R. & Zhuang, X. RNA imaging with multiplexed error-robust fluorescence in situ hybridization (MERFISH). Methods Enzymol. 572, 1–49 (2016).
Dean, K.M., Roudot, P., Welf, E.S., Danuser, G. & Fiolka, R. Deconvolution-free subcellular imaging with axially swept light sheet microscopy. Biophys. J. 108, 2807–2815 (2015).
Hedde, P.N. & Gratton, E. Selective plane illumination microscopy with a light sheet of uniform thickness formed by an electrically tunable lens. Microsc. Res. Tech. 6, 2181 (2016).
Oh, S.W. et al. A mesoscale connectome of the mouse brain. Nature 508, 207–214 (2014).
Schnell, S.A., Staines, W.A. & Wessendorf, M.W. Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J. Histochem. Cytochem. 47, 719–730 (1999).
Gómez-Gaviro, M.V. et al. Optimized CUBIC protocol for three-dimensional imaging of chicken embryos at single-cell resolution. Development 144, 2092–2097 (2017).
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.
K.C. is a co-inventor on patent application owned by MIT covering the SHIELD and SWITCH technology (PCT/US2016/064538).
Integrated supplementary information
(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: brain-map.org/api/index.html). (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.
(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.
(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).
Supplementary Figures 1–5 (PDF 1678 kb)
Supplementary Tables 1–3 and Supplementary Note (PDF 538 kb)
A series of xy-plane images showing uniform (dT)50-Cy3 FISH signal in a SHIELD-processed block. (MP4 19074 kb)
3D autofluorescence (white) and YFP (green) signals from Thy1-H line mouse hemispheres processed with PACT, iDISCO+ and SHIELD. (MP4 22203 kb)
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)
3D rendering of GFAP (green) and CR (red) immunolabeling of a 1-mm-thick SHIELD-processed human cortical coronal block. (MP4 104641 kb)
3D rendering showing a fresh biopsy sample processed with the SHIELD-based rapid (<4 h) phenotyping pipeline. (MP4 13714 kb)
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)
A series of optical sections of SHIELD-MAP tissue from the surface to a depth of 5 mm. (MP4 122726 kb)
3D rendering of a reconstructed YFP-expressing neuron in a Thy1-YFP H line mouse brain cortex using SHIELD-MAP. (MP4 130548 kb)
3D rendering of hemisphere showing labeled GPe-PV+ circuitry. (MP4 203075 kb)
3D rendering of 1-mm-thick mouse tissue showing labeled GPe-PV+ circuitry after first-round GAD1-FISH (cyan) staining. (MP4 112693 kb)
3D super-resolution images of putative axosomatic connectivity. (MP4 71545 kb)
About this article
Cite this article
Park, Y., Sohn, C., Chen, R. et al. Protection of tissue physicochemical properties using polyfunctional crosslinkers. Nat Biotechnol 37, 73–83 (2019). https://doi.org/10.1038/nbt.4281
Nature Reviews Neuroscience (2020)
Journal of Neuroscience Methods (2020)
Nature Protocols (2019)