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A highly homogeneous polymer composed of tetrahedron-like monomers for high-isotropy expansion microscopy


Expansion microscopy (ExM) physically magnifies biological specimens to enable nanoscale-resolution imaging using conventional microscopes. Current ExM methods permeate specimens with free-radical-chain-growth-polymerized polyacrylate hydrogels, whose network structure limits the local isotropy of expansion as well as the preservation of morphology and shape at the nanoscale. Here we report that ExM is possible using hydrogels that have a more homogeneous network structure, assembled via non-radical terminal linking of tetrahedral monomers. As with earlier forms of ExM, such ‘tetra-gel’-embedded specimens can be iteratively expanded for greater physical magnification. Iterative tetra-gel expansion of herpes simplex virus type 1 (HSV-1) virions by ~10× in linear dimension results in a median spatial error of 9.2 nm for localizing the viral envelope layer, rather than 14.3 nm from earlier versions of ExM. Moreover, tetra-gel-based expansion better preserves the virion spherical shape. Thus, tetra-gels may support ExM with reduced spatial errors and improved local isotropy, pointing the way towards single-biomolecule accuracy ExM.

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Fig. 1: Design and synthesis of the tetra-gel (TG) for ExM.
Fig. 2: TG-mediated expansion of cells and tissues.
Fig. 3: TG-based iterative expansion.
Fig. 4: Spatial errors introduced by TG-based versus classical PAAG-based iterative expansion.
Fig. 5: Shape analysis of TG- versus PAAG-expanded HSV-1 virions.

Data availability

Source data are provided with this paper. The total raw data size of the study exceeds 350 GB. The data that support this study are available from the authors upon reasonable request.

Code availability

Analysis code used in this study, including virus particle analysis48, microtubule peak-to-peak distance analysis49 and HSV-1 averaged particle image simulation50 are available at


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We thank Y.-Y. Chou and T. Kirchhausen at HMS for help with VSV stock preparation and virion immobilization; C. Linghu and O. Shemesh for help with HSV-1 stock preparation; P. Valdes and C. Zhang for helpful discussion about sample staining and expansion; M. J. Kauke for helpful discussion about DNA staining; S. M. Asano for helpful discussion about image analysis; G. H. Huynh for help with mouse brain slice preparation; F. Chen for helpful discussion about monomer and polymerization chemistry design; R. Herlo at HMS for helpful discussion about virion envelope proteins. E.S.B. acknowledges L. Yang and Y. E. Tan, J. Doerr, the Open Philanthropy Project, NIH 1R01NS087950, NIH 1RM1HG008525, NIH 1R01DA045549, NIH 2R01DA029639, NIH 1R01NS102727, NIH 1R01EB024261, NIH 1R01MH110932, the HHMI-Simons Faculty Scholars Program, the HHMI Investigator program, IARPA D16PC00008, the US Army Research Laboratory and the US Army Research Office under contract/grant W911NF1510548, the US–Israel Binational Science Foundation Grant 2014509, NSF Grant 1734870 and the NIH Director’s Pioneer Award 1DP1NS087724. C.-C.Y. acknowledges the McGovern Institute for Brain Research at MIT for the Friends of the McGovern Fellowship. S.U. was supported by Biogen and NIH 5R01GM075252-13S grants awarded to T. Kirchhausen, and acknowledges support from Philomathia Foundation and Chan Zuckerberg Initiative Imaging Scientist program.

Author information

Authors and Affiliations



R.G. and L.G. designed and synthesized the monomers and conducted initial gelation experiments. C.-C.Y. and R.G. designed and conducted iterative expansion, virion expansion and associated analysis. C.-C.Y. created the semi-automated virion analysis pipeline and the simulation model. K.D.P. helped characterization of the gel in cell culture. R.L.N. purified HSV-1 and prepared the virion stock solution. J.B.M. provided purified HIV virions. S.U. provided purified VSV virions and conducted initial virion immobilization experiments. C.-C.Y., R.G. and L.G. processed and performed quantitative analysis of all image data. R.G., C.-C.Y. and E.S.B. wrote the manuscript with input from all co-authors. E.S.B. supervised the project.

Corresponding author

Correspondence to Edward S. Boyden.

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Competing interests

R.G., C.-C.Y., L.G. and E.S.B. have filed for patent protection on a subset of the technologies here described. E.S.B. is a cofounder of a company that aims to commercialize ExM for medical purposes. R.G., C.-C.Y., L.G. and E.S.B. are co-inventors on multiple patents related to ExM. The authors declare no other competing interests.

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Supplementary information

Supplementary Information

Supplementary methods, Figs. 1–8 and Tables 1–3.

Reporting Summary


Supplementary Video 1 Three-dimensional (3D) rendered movie of envelope proteins of a herpes simplex virus type 1 (HSV-1) virion expanded by tetra-gel (TG)-based three-round iterative expansion. The deconvolved puncta (white), the overlay of the deconvolved puncta (white) and the fitted centroids (red), and the extracted centroids (red) are shown from left to right. Expansion factor, 38.3×. Scale bars, 100 nm (3.83 µm). The featured virion corresponds to virion b in Supplementary Fig. 8.

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Numerical data used to generate graphs.

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Gao, R., Yu, CC.(., Gao, L. et al. A highly homogeneous polymer composed of tetrahedron-like monomers for high-isotropy expansion microscopy. Nat. Nanotechnol. 16, 698–707 (2021).

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