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Nanoscale imaging of clinical specimens using conventional and rapid-expansion pathology

Abstract

In pathology, microscopy is an important tool for the analysis of human tissues, both for the scientific study of disease states and for diagnosis. However, the microscopes commonly used in pathology are limited in resolution by diffraction. Recently, we discovered that it was possible, through a chemical process, to isotropically expand preserved cells and tissues by 4–5× in linear dimension. We call this process expansion microscopy (ExM). ExM enables nanoscale resolution imaging on conventional microscopes. Here we describe protocols for the simple and effective physical expansion of a variety of human tissues and clinical specimens, including paraffin-embedded, fresh frozen and chemically stained human tissues. These protocols require only inexpensive, commercially available reagents and hardware commonly found in a routine pathology laboratory. Our protocols are written for researchers and pathologists experienced in conventional fluorescence microscopy. The conventional protocol, expansion pathology, can be completed in ~1 d with immunostained tissue sections and 2 d with unstained specimens. We also include a new, fast variant, rapid expansion pathology, that can be performed on <5-µm-thick tissue sections, taking <4 h with immunostained tissue sections and <8 h with unstained specimens.

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Fig. 1: Workflows of conventional (ExPath) and rapid (rExPath) expansion pathology.
Fig. 2: Validation of conventional (ExPath) and rapid (rExPath) expansion pathology.
Fig. 3: Comparison of ExPath and rExPath on adjacent human prostate FFPE tissue sections.
Fig. 4: ExPath reduction of tissue autofluorescence.
Fig. 5: ExPath imaging of a wide range of human tissue types.
Fig. 6: Rapid ExPath imaging of lymph node specimens from patients.
Fig. 7: ExPath and rExPath imaging of H&E-stained tissue sections and frozen tissue sections.
Fig. 8: Gelling station setup and specimen pre-treatment before proteinase K digestion.

Data availability

Part of the primary data underlying the figures presented in this article can be found as examples in https://github.com/zhao-biophotonics/ExPath-reg; the rest of the primary data can be provided upon reasonable request from the corresponding authors.

References

  1. 1.

    Hell, S. W. Far-field optical nanoscopy. In 2010 23rd Annual Meeting of the IEEE Photonics Society, PHOTINICS 2010. 3–4 (IEEE, 2010) https://doi.org/10.1109/PHOTONICS.2010.5698725

  2. 2.

    Zhuang, X. Nano-imaging with STORM. Nat. Photonics 3, 365–367 (2009).

    CAS  Article  Google Scholar 

  3. 3.

    Huang, B., Bates, M. & Zhuang, X. Super-resolution fluorescence microscopy. Annu. Rev. Biochem. 78, 993–1016 (2009).

    CAS  Article  Google Scholar 

  4. 4.

    Betzig, E. Proposed method for molecular optical imaging. Opt. Lett. 20, 237–239 (1995).

    CAS  Article  Google Scholar 

  5. 5.

    Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    CAS  Article  Google Scholar 

  6. 6.

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

    CAS  Article  Google Scholar 

  7. 7.

    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  Article  Google Scholar 

  8. 8.

    Zhao, Y. et al. Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy. Nat. Biotechnol. 35, 757–764 (2017).

    CAS  Article  Google Scholar 

  9. 9.

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

    CAS  Article  Google Scholar 

  10. 10.

    Hausen, P. & Dreyer, C. The use of polyacrylamide as an embedding medium for immunohistochemical studies of embryonic tissues. Biotech. Histochem. 56, 287–293 (1981).

    CAS  Google Scholar 

  11. 11.

    Wassie, A. T., Zhao, Y. & Boyden, E. S. Expansion microscopy: principles and uses in biological research. Nat. Methods 16, 33–41 (2019).

    CAS  Article  Google Scholar 

  12. 12.

    Chozinski, T. J. et al. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat. Methods 13, 1–7 (2016).

    Article  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  Article  Google Scholar 

  14. 14.

    Chang, J.-B. et al. Iterative expansion microscopy. Nat. Methods 14, 593–599 (2017).

    CAS  Article  Google Scholar 

  15. 15.

    Asano, S. M. et al. Expansion microscopy: protocols for imaging proteins and RNA in cells and tissues. Curr. Protoc. Cell Biol. 80, e56 (2018).

    Article  Google Scholar 

  16. 16.

    Karagiannis, E. & Boyden, E. Expansion microscopy: development and neuroscience applications. Curr. Opin. Neurobiol. 50, 56–63 (2018).

    CAS  Article  Google Scholar 

  17. 17.

    Gao, R., Asano, S. M. & Boyden, E. S. Q&A: expansion microscopy. BMC Biol. 15, 50 (2017).

    Article  Google Scholar 

  18. 18.

    Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

    CAS  Article  Google Scholar 

  19. 19.

    Klar, T. A., Jakobs, S., Dyba, M., Egner, A. & Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000).

    CAS  Article  Google Scholar 

  20. 20.

    Gustafsson, M. G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

    CAS  Article  Google Scholar 

  21. 21.

    Heintzmann, R. & Cremer, C. G. Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. In SPIE Proceedings Vol. 3568: Optical Biopsies and Microscopic Techniques III (eds Bigio, I. J., Schneckenburger, H., Slavik, J., Svanberg, K. & Viallet, P. M.) 185–196 (International Society for Optics and Photonics, 1999) https://spie.org/Publications/Proceedings/Paper/10.1117/12.336833

  22. 22.

    Rust, M. J., Bates, M. & Zhuang, X. W. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    CAS  Article  Google Scholar 

  23. 23.

    Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014).

    CAS  Article  Google Scholar 

  24. 24.

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

    Article  Google Scholar 

  25. 25.

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

    CAS  Article  Google Scholar 

  26. 26.

    Dodt, H.-U. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4, 331–336 (2007).

    CAS  Article  Google Scholar 

  27. 27.

    Ke, M.-T., Fujimoto, S. & Imai, T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat. Neurosci. 16, 1154–1161 (2013).

    CAS  Article  Google Scholar 

  28. 28.

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

    CAS  Article  Google Scholar 

  29. 29.

    Susaki, E. A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014).

    CAS  Article  Google Scholar 

  30. 30.

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

    CAS  Article  Google Scholar 

  31. 31.

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

    CAS  Article  Google Scholar 

  32. 32.

    Gao, R. et al. Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution. Science 363, eaau8302 (2019).

    CAS  Article  Google Scholar 

  33. 33.

    Halpern, A. R., Alas, G. C. M., Chozinski, T. J., Paredez, A. R. & Vaughan, J. C. Hybrid structured illumination expansion microscopy reveals microbial cytoskeleton organization. ACS Nano 11, 12677–12686 (2017).

    CAS  Article  Google Scholar 

  34. 34.

    Wang, Y. et al. Combined expansion microscopy with structured illumination microscopy for analyzing protein complexes. Nat. Protoc. 13, 1869–1895 (2018).

    CAS  Article  Google Scholar 

  35. 35.

    Cahoon, C. K. et al. Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex. Proc. Natl Acad. Sci. USA 114, E6857–E6866 (2017).

    CAS  Article  Google Scholar 

  36. 36.

    Tong, Z. et al. Ex-STORM: expansion single molecule super-resolution microscopy. Preprint at https://www.biorxiv.org/content/10.1101/049403v2 (2016).

  37. 37.

    Gao, M. et al. Expansion stimulated emission depletion microscopy (ExSTED). ACS Nano 12, 4178–4185 (2018).

    CAS  Article  Google Scholar 

  38. 38.

    Gambarotto, D. et al. Imaging cellular ultrastructures using expansion microscopy (U-ExM). Nat. Methods 16, 71–74 (2019).

    CAS  Article  Google Scholar 

  39. 39.

    Truckenbrodt, S., Sommer, C., Rizzoli, S. O. & Danzl, J. G. A practical guide to optimization in X10 expansion microscopy. Nat. Protoc. 14, 832–863 (2019).

    CAS  Article  Google Scholar 

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Acknowledgements

Human lymph node specimens were from the pathology archives of the Harvard University Center for AIDS Research, obtained under IRB protocol #2010P000632 to B.D.W. For funding, E.S.B. acknowledges L. Yang, Schmidt Futures, the MIT Media Lab, the Chan Zuckerberg Initiative, NIH U01MH114819, NIH 1U19MH114821, the Ludwig Foundation, NIH 1R01NS102727, John Doerr, NIH 1R01EB024261, the Open Philanthropy project, the HHMI-Simons Faculty Scholars Program, the US Army Research Laboratory and the US Army Research Office under contract/grant number W911NF1510548, NIH 1R01MH110932 and NIH 1RM1HG008525. O.B. acknowledges support from the Ludwig Center at Harvard and from Harvard Catalyst (the Harvard Clinical and Translational Science Center (National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health Award UL1 TR001102)). Y.Z. acknowledges support from Carnegie Mellon University and NIH Director’s New Innovator Award (DP2 OD025926-01).

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Authors

Contributions

Y.Z., O.B. and E.S.B. wrote the manuscript. Y.Z., O.B. and F.F. conducted the experiments. Y.Z., O.B., F.F. and E.S.B. analyzed the data. M.C. and S.W. conducted STED imaging for validation of rExPath. J.D. filmed and photographed the ExPath process. G.H.M., N.L.L. and B.D.W. provided de-identified human clinical specimens and helped with the experiments. Y.Z. and E.S.B. oversaw the research.

Corresponding authors

Correspondence to Edward S. Boyden or Yongxin Zhao.

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Competing interests

The authors have filed and obtained patent protection on a subset of the technologies here described (US provisional application no. 62/299,754, 62/463,265 and 62/463,251). E.S.B. helped cofound a company to help disseminate ExM to the community. O.B. is the Co-Founder and CEO of QPathology LLC, Boston, MA.

Additional information

Peer review information Nature Protocols thanks Sven Truckenbrodt and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Zhao, Y. et al. Nat. Biotechnol. 35, 757–764 (2017): https://doi.org/10.1038/nbt.3892

Gao, R. et al. Science 363, eaau8302 (2019): https://doi.org/10.1126/science.aau8302

Wassie, A. T. et al. Nat. Methods 16, 33–41 (2019): https://doi.org/10.1038/s41592-018-0219-4

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Bucur, O., Fu, F., Calderon, M. et al. Nanoscale imaging of clinical specimens using conventional and rapid-expansion pathology. Nat Protoc 15, 1649–1672 (2020). https://doi.org/10.1038/s41596-020-0300-1

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