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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  1. 1

    Masland, R.H. Neuronal cell types. Curr. Biol. 14, R497–R500 (2004).

    CAS  PubMed  Google Scholar 

  2. 2

    Kepecs, A. & Fishell, G. Interneuron cell types are fit to function. Nature 505, 318–326 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Alivisatos, A.P. et al. The brain activity map. Science 339, 1284–1285 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    DeFelipe, J. From the connectome to the synaptome: an epic love story. Science 330, 1198–1201 (2010).

    CAS  PubMed  Google Scholar 

  5. 5

    Zingg, B. et al. Neural networks of the mouse neocortex. Cell 156, 1096–1111 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Kasthuri, N. et al. Saturated reconstruction of a volume of neocortex. Cell 162, 648–661 (2015).

    CAS  PubMed  Google Scholar 

  7. 7

    Crosetto, N., Bienko, M. & van Oudenaarden, A. Spatially resolved transcriptomics and beyond. Nat. Rev. Genet. 16, 57–66 (2015).

    CAS  PubMed  Google Scholar 

  8. 8

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Giesen, C. et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat. Methods 11, 417–422 (2014).

    CAS  PubMed  Google Scholar 

  10. 10

    Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332–337 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    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).

    CAS  PubMed  Google Scholar 

  12. 12

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Murray, E. et al. Simple, scalable proteomic imaging for high-dimensional profiling of intact systems. Cell 163, 1500–1514 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Renier, N. et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 (2014).

    CAS  PubMed  Google Scholar 

  16. 16

    Pan, C. et al. Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat. Methods 13, 859–867 (2016).

    CAS  PubMed  Google Scholar 

  17. 17

    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).

    CAS  PubMed  Google Scholar 

  18. 18

    Ertürk, A. et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat. Protoc. 7, 1983–1995 (2012).

    PubMed  Google Scholar 

  19. 19

    Chen, F., Tillberg, P.W. & Boyden, E.S. Expansion microscopy. Science 347, 543–548 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    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).

    CAS  PubMed  Google Scholar 

  21. 21

    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).

    CAS  PubMed  Google Scholar 

  22. 22

    Kabanov, A.V. & Vinogradov, S.V. Nanogels as pharmaceutical carriers: finite networks of infinite capabilities. Angew. Chem. Int. Ed. Engl. 48, 5418–5429 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Sheldon, R.A. & van Pelt, S. Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soc. Rev. 42, 6223–6235 (2013).

    CAS  PubMed  Google Scholar 

  24. 24

    Hartmann, M. Ordered mesoporous materials for bioadsorption and biocatalysis. Chem. Mater. 17, 4577–4593 (2005).

    CAS  Google Scholar 

  25. 25

    Mateo, C. et al. Some special features of glyoxyl supports to immobilize proteins. Enzyme Microb. Technol. 37, 456–462 (2005).

    CAS  Google Scholar 

  26. 26

    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).

    CAS  PubMed  Google Scholar 

  27. 27

    Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    CAS  PubMed  Google Scholar 

  28. 28

    Kim, S.-Y. et al. Stochastic electrotransport selectively enhances the transport of highly electromobile molecules. Proc. Natl. Acad. Sci. USA 112, E6274–E6283 (2015).

    CAS  PubMed  Google Scholar 

  29. 29

    Tsien, R.Y. Fluorescent probes of cell signaling. Annu. Rev. Neurosci. 12, 227–253 (1989).

    CAS  PubMed  Google Scholar 

  30. 30

    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).

    CAS  PubMed  Google Scholar 

  31. 31

    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).

    CAS  PubMed  Google Scholar 

  32. 32

    Enoki, S., Saeki, K., Maki, K. & Kuwajima, K. Acid denaturation and refolding of green fluorescent protein. Biochemistry 43, 14238–14248 (2004).

    CAS  PubMed  Google Scholar 

  33. 33

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Hama, H. et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci. 14, 1481–1488 (2011).

    CAS  PubMed  Google Scholar 

  35. 35

    Hopwood, D. Theoretical and practical aspects of glutaraldehyde fixation. Histochem. J. 4, 267–303 (1972).

    CAS  PubMed  Google Scholar 

  36. 36

    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).

    CAS  Google Scholar 

  37. 37

    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).

    CAS  PubMed  Google Scholar 

  38. 38

    Pena, J.T.G. et al. miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat. Methods 6, 139–141 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    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).

    CAS  PubMed  Google Scholar 

  40. 40

    Nojima, S. et al. CUBIC pathology: three-dimensional imaging for pathological diagnosis. Sci. Rep. 7, 9269 (2017).

    PubMed  PubMed Central  Google Scholar 

  41. 41

    Tanaka, N. et al. Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity. Nat. Biomed. Eng. 1, 796–806 (2017).

    CAS  PubMed  Google Scholar 

  42. 42

    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).

    Google Scholar 

  43. 43

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Ke, M.-T. Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent. Cell Rep. 14, 2718–2732 (2016).

    CAS  PubMed  Google Scholar 

  46. 46

    Gerfen, C.R. & Wilson, C.J. in Handbook of Chemical Neuroanatomy 12, 371–468 (Elsevier, 1996).

    Google Scholar 

  47. 47

    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).

    PubMed  Google Scholar 

  48. 48

    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).

    PubMed  PubMed Central  Google Scholar 

  49. 49

    Knowland, D. et al. Distinct ventral pallidal neural populations mediate separate symptoms of depression. Cell 170, 284–297.e18 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Ye, L. et al. Wiring and molecular features of prefrontal ensembles representing distinct experiences. Cell 165, 1776–1788 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Economo, M.N. et al. A platform for brain-wide imaging and reconstruction of individual neurons. Elife 5, e10566 (2016).

    PubMed  PubMed Central  Google Scholar 

  52. 52

    Luo, L., Callaway, E.M. & Svoboda, K. Genetic dissection of neural circuits: a decade of progress. Neuron 98, 256–281 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    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).

    PubMed  PubMed Central  Google Scholar 

  54. 54

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Spurr, A.R. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31–43 (1969).

    CAS  PubMed  Google Scholar 

  56. 56

    Halgren, T.A. Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94. J. Comput. Chem. 17, 490–519 (1996).

    CAS  Google Scholar 

  57. 57

    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).

    CAS  Google Scholar 

  58. 58

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    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).

    CAS  Google Scholar 

  60. 60

    Yang, B. et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158, 945–958 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Renier, N. et al. Mapping of brain activity by automated volume analysis of immediate early genes. Cell 165, 1789–1802 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63

    Chen, F. et al. Nanoscale imaging of RNA with expansion microscopy. Nat. Methods 13, 679–684 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65

    Moffitt, J.R. & Zhuang, X. RNA imaging with multiplexed error-robust fluorescence in situ hybridization (MERFISH). Methods Enzymol. 572, 1–49 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67

    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).

    Google Scholar 

  68. 68

    Oh, S.W. et al. A mesoscale connectome of the mouse brain. Nature 508, 207–214 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69

    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).

    CAS  PubMed  Google Scholar 

  70. 70

    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).

    PubMed  Google Scholar 

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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.

Author information




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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