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


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.

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Part of the primary data underlying the figures presented in this article can be found as examples in; the rest of the primary data can be provided upon reasonable request from the corresponding authors.


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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 and Affiliations



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.

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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):

Gao, R. et al. Science 363, eaau8302 (2019):

Wassie, A. T. et al. Nat. Methods 16, 33–41 (2019):

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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).

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