Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Protein Data Bank

References

  1. 1.

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

  2. 2.

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

  3. 3.

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

  4. 4.

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

  5. 5.

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

  6. 6.

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

  7. 7.

    , & Spatially resolved transcriptomics and beyond. Nat. Rev. Genet. 16, 57–66 (2015).

  8. 8.

    , , & In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus. Neuron 92, 342–357 (2016).

  9. 9.

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

  10. 10.

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

  11. 11.

    & 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).

  12. 12.

    , , , & Multiplexed intact-tissue transcriptional analysis at cellular resolution. Cell 164, 792–804 (2016).

  13. 13.

    et al. Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues. Nat. Biotechnol. 34, 973–981 (2016).

  14. 14.

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

  15. 15.

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

  16. 16.

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

  17. 17.

    , , , & Chemical principles in tissue clearing and staining protocols for whole-body cell profiling. Annu. Rev. Cell Dev. Biol. 32, 713–741 (2016).

  18. 18.

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

  19. 19.

    , & Expansion microscopy. Science 347, 543–548 (2015).

  20. 20.

    , , & A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples. Lab. Invest. 83, 1427–1435 (2003).

  21. 21.

    , & Therapeutic protein-polymer conjugates: advancing beyond PEGylation. J. Am. Chem. Soc. 136, 14323–14332 (2014).

  22. 22.

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

  23. 23.

    & Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soc. Rev. 42, 6223–6235 (2013).

  24. 24.

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

  25. 25.

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

  26. 26.

    , , & Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. Biotechniques 37, 790–796, 798–802 (2004).

  27. 27.

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

  28. 28.

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

  29. 29.

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

  30. 30.

    , & Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry 18, 5191–5196 (1979).

  31. 31.

    & Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation. J. Histochem. Cytochem. 39, 225–229 (1991).

  32. 32.

    , , & Acid denaturation and refolding of green fluorescent protein. Biochemistry 43, 14238–14248 (2004).

  33. 33.

    , , & Antigen retrieval causes protein unfolding: evidence for a linear epitope model of recovered immunoreactivity. J. Histochem. Cytochem. 59, 366–381 (2011).

  34. 34.

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

  35. 35.

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

  36. 36.

    , , , & Autofluorescence generation and elimination: a lesson from glutaraldehyde. Chem. Commun. (Camb.) 49, 3028–3030 (2013).

  37. 37.

    , , , & Analysis of DNA adducts in DNA hydrolysates by capillary zone electrophoresis and capillary zone electrophoresis-electrospray mass spectrometry. Anal. Chem. 68, 3575–3584 (1996).

  38. 38.

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

  39. 39.

    et al. Role of image-guided core-needle biopsy in the management of patients with lymphoma. J. Clin. Oncol. 14, 2427–2430 (1996).

  40. 40.

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

  41. 41.

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

  42. 42.

    et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat. Biomed. Eng. 1, 1–10 (2017).

  43. 43.

    et al. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat. Biotechnol. 34, 987–992 (2016).

  44. 44.

    , & High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy. Nat. Protoc. 6, 1391–1411 (2011).

  45. 45.

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

  46. 46.

    & in Handbook of Chemical Neuroanatomy 12, 371–468 (Elsevier, 1996).

  47. 47.

    , , , & The expanding universe of disorders of the basal ganglia. Lancet 384, 523–531 (2014).

  48. 48.

    , & Globus pallidus externus neurons expressing parvalbumin interconnect the subthalamic nucleus and striatal interneurons. PLoS One 11, e0149798 (2016).

  49. 49.

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

  50. 50.

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

  51. 51.

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

  52. 52.

    , & Genetic dissection of neural circuits: a decade of progress. Neuron 98, 256–281 (2018).

  53. 53.

    , , , & Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).

  54. 54.

    et al. Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat. Commun. 6, 7147 (2015).

  55. 55.

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

  56. 56.

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

  57. 57.

    Merck molecular force field. II. MMFF94 van der Waals and electrostatic parameters for intermolecular interactions. J. Comput. Chem. 17, 520–552 (1996).

  58. 58.

    , & Crystal structure of enhanced green fluorescent protein to 1.35 Å resolution reveals alternative conformations for Glu222. PLoS One 7, e47132 (2012).

  59. 59.

    , & 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).

  60. 60.

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

  61. 61.

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

  62. 62.

    , & Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability. ACS Nano 8, 4284–4294 (2014).

  63. 63.

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

  64. 64.

    et al. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing. Development 143, 2862–2867 (2016).

  65. 65.

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

  66. 66.

    , , , & Deconvolution-free subcellular imaging with axially swept light sheet microscopy. Biophys. J. 108, 2807–2815 (2015).

  67. 67.

    & Selective plane illumination microscopy with a light sheet of uniform thickness formed by an electrically tunable lens. Microsc. Res. Tech. 6, 2181 (2016).

  68. 68.

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

  69. 69.

    , & Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J. Histochem. Cytochem. 47, 719–730 (1999).

  70. 70.

    et al. Optimized CUBIC protocol for three-dimensional imaging of chicken embryos at single-cell resolution. Development 144, 2092–2097 (2017).

Download references

Acknowledgements

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

Author notes

    • Young-Gyun Park
    • , Chang Ho Sohn
    •  & Ritchie Chen

    These authors contributed equally to this work.

Affiliations

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

    • Young-Gyun Park
    • , Chang Ho Sohn
    • , Ritchie Chen
    • , Margaret McCue
    • , Dae Hee Yun
    • , Gabrielle T Drummond
    • , Taeyun Ku
    • , Nicholas B Evans
    • , Heejin Choi
    • , Xin Jin
    •  & Kwanghun Chung
  2. Picower Institute for Learning and Memory, MIT, Cambridge, Massachusetts, USA.

    • Young-Gyun Park
    • , Chang Ho Sohn
    • , Ritchie Chen
    • , Margaret McCue
    • , Dae Hee Yun
    • , Gabrielle T Drummond
    • , Taeyun Ku
    • , Nicholas B Evans
    • , Heejin Choi
    •  & Kwanghun Chung
  3. Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA.

    • Hayeon Caitlyn Oak
    • , Wendy Trieu
    •  & Kwanghun Chung
  4. Broad Institute of Harvard University and MIT, Cambridge, Massachusetts, USA.

    • Xin Jin
    • , Todd R Golub
    •  & Kwanghun Chung
  5. Neurobiology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, California, USA.

    • Varoth Lilascharoen
    •  & Byung Kook Lim
  6. Department of Mechanical and Automation Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong.

    • Ji Wang
    •  & Shih-Chi Chen
  7. Program in Cellular and Molecular Medicine, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

    • Matthias C Truttmann
  8. Department of Chemical Engineering, MIT, Cambridge, Massachusetts, USA.

    • Helena W Qi
    • , Heather J Kulik
    •  & Kwanghun Chung
  9. Department of Chemistry, MIT, Cambridge, Massachusetts, USA.

    • Helena W Qi
  10. Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

    • Hidde L Ploegh
  11. C.S. Kubik Laboratory for Neuropathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.

    • Matthew P Frosch

Authors

  1. Search for Young-Gyun Park in:

  2. Search for Chang Ho Sohn in:

  3. Search for Ritchie Chen in:

  4. Search for Margaret McCue in:

  5. Search for Dae Hee Yun in:

  6. Search for Gabrielle T Drummond in:

  7. Search for Taeyun Ku in:

  8. Search for Nicholas B Evans in:

  9. Search for Hayeon Caitlyn Oak in:

  10. Search for Wendy Trieu in:

  11. Search for Heejin Choi in:

  12. Search for Xin Jin in:

  13. Search for Varoth Lilascharoen in:

  14. Search for Ji Wang in:

  15. Search for Matthias C Truttmann in:

  16. Search for Helena W Qi in:

  17. Search for Hidde L Ploegh in:

  18. Search for Todd R Golub in:

  19. Search for Shih-Chi Chen in:

  20. Search for Matthew P Frosch in:

  21. Search for Heather J Kulik in:

  22. Search for Byung Kook Lim in:

  23. Search for Kwanghun Chung in:

Contributions

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.

Competing interests

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

Corresponding author

Correspondence to Kwanghun Chung.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Tables and Note

    Supplementary Tables 1–3 and Supplementary Note

Videos

  1. 1.

    Supplementary Video 1

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

  2. 2.

    Supplementary Video 2

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

  3. 3.

    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).

  4. 4.

    Supplementary Video 4

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

  5. 5.

    Supplementary Video 5

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

  6. 6.

    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).

  7. 7.

    Supplementary Video 7

    A series of optical sections of SHIELD-MAP tissue from the surface to a depth of 5 mm.

  8. 8.

    Supplementary Video 8

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

  9. 9.

    Supplementary Video 9

    3D rendering of hemisphere showing labeled GPe-PV+ circuitry.

  10. 10.

    Supplementary Video 10

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

  11. 11.

    Supplementary Video 11

    3D super-resolution images of putative axosomatic connectivity.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt.4281