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Expansion microscopy: principles and uses in biological research


Many biological investigations require 3D imaging of cells or tissues with nanoscale spatial resolution. We recently discovered that preserved biological specimens can be physically expanded in an isotropic fashion through a chemical process. Expansion microscopy (ExM) allows nanoscale imaging of biological specimens with conventional microscopes, decrowds biomolecules in support of signal amplification and multiplexed readout chemistries, and makes specimens transparent. We review the principles of how ExM works, advances in the technology made by our group and others, and its applications throughout biology and medicine.

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Fig. 1: ExM concept and example outcomes.
Fig. 2: Applications of ExM in biology and medicine.
Fig. 3: ExM decrowds biomolecules, facilitating post-expansion staining, signal amplification, and multiplexed readout.
Fig. 4: Results of 25-nm-resolution imaging of neural circuitry and synapses with iExM.


  1. 1.

    Dunn, R. C. Near-field scanning optical microscopy. Chem. Rev. 99, 2891–2928 (1999).

    CAS  Article  Google Scholar 

  2. 2.

    Dürig, U., Pohl, D. W. & Rohner, F. Near-field optical-scanning microscopy. J. Appl. Phys. 59, 3318–3327 (1986).

    Article  Google Scholar 

  3. 3.

    Hell, S.W. Far-field optical nanoscopy. In Proc. 2010 23rd Annual Meeting of the IEEE Photonics Society (eds Jagadish, C. et al.) 3–4 (IEEE, New York, 2010).

  4. 4.

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

    CAS  Article  Google Scholar 

  5. 5.

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

    CAS  Article  Google Scholar 

  6. 6.

    Tanaka, T. et al. Phase transitions in ionic gels. Phys. Rev. Lett. 45, 1636–1639 (1980).

    CAS  Article  Google Scholar 

  7. 7.

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

    CAS  Article  Google Scholar 

  8. 8.

    Cohen, Y., Ramon, O., Kopelman, I. J. & Mizrahi, S. Characterization of inhomogeneous polyacrylamide hydrogels. J. Polym. Sci. B Polym. Phys. 30, 1055–1067 (1992).

    CAS  Article  Google Scholar 

  9. 9.

    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 

  10. 10.

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

    CAS  Article  Google Scholar 

  11. 11.

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

    CAS  Article  Google Scholar 

  12. 12.

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

    CAS  Article  Google Scholar 

  13. 13.

    Chozinski, T. J. et al. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat. Methods 13, 485–488 (2016).

    CAS  Article  Google Scholar 

  14. 14.

    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 

  15. 15.

    Truckenbrodt, S. et al. X10 expansion microscopy enables 25-nm resolution on conventional microscopes. EMBO Rep. 19, e45836 (2018).

    Article  Google Scholar 

  16. 16.

    Tsanov, N. et al. smiFISH and FISH-quant—a flexible single RNA detection approach with super-resolution capability. Nucleic Acids Res. 44, e165 (2016).

    Article  Google Scholar 

  17. 17.

    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 

  18. 18.

    Freifeld, L. et al. Expansion microscopy of zebrafish for neuroscience and developmental biology studies. Proc. Natl Acad. Sci. USA 114, E10799–E10808 (2017).

    CAS  Article  Google Scholar 

  19. 19.

    Migliori, B. et al. Light sheet theta microscopy for rapid high-resolution imaging of large biological samples. BMC Biol. 16, 57 (2018).

    Article  Google Scholar 

  20. 20.

    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 

  21. 21.

    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 

  22. 22.

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

    CAS  Article  Google Scholar 

  23. 23.

    Murakami, T. C. et al. A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing. Nat. Neurosci. 21, 625–637 (2018).

    CAS  Article  Google Scholar 

  24. 24.

    Zhang, Y. S. et al. Hybrid microscopy: enabling inexpensive high-performance imaging through combined physical and optical magnifications. Sci. Rep. 6, 22691 (2016).

    CAS  Article  Google Scholar 

  25. 25.

    Aoki, T., Tsuchida, S., Yahara, T. & Hamaue, N. Novel assays for proteases using green fluorescent protein-tagged substrate immobilized on a membrane disk. Anal. Biochem. 378, 132–137 (2008).

    CAS  Article  Google Scholar 

  26. 26.

    Nicholls, S. B. & Hardy, J. A. Structural basis of fluorescence quenching in caspase activatable-GFP. Protein Sci. 22, 247–257 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Deshpande, T. et al. Subcellular reorganization and altered phosphorylation of the astrocytic gap junction protein connexin43 in human and experimental temporal lobe epilepsy. Glia 65, 1809–1820 (2017).

    Article  Google Scholar 

  28. 28.

    Crittenden, J. R. et al. Striosome-dendron bouquets highlight a unique striatonigral circuit targeting dopamine-containing neurons. Proc. Natl Acad. Sci. USA 113, 11318–11323 (2016).

    CAS  Article  Google Scholar 

  29. 29.

    Decarreau, J. et al. The tetrameric kinesin Kif25 suppresses pre-mitotic centrosome separation to establish proper spindle orientation. Nat. Cell Biol. 19, 384–390 (2017).

    CAS  Article  Google Scholar 

  30. 30.

    Suofu, Y. et al. Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release. Proc. Natl Acad. Sci. USA 114, E7997–E8006 (2017).

    CAS  Article  Google Scholar 

  31. 31.

    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 

  32. 32.

    Orth, A. et al. Super-multiplexed fluorescence microscopy via photostability contrast. Biomed. Opt. Express 9, 2943–2954 (2018).

    Article  Google Scholar 

  33. 33.

    Chozinski, T. J. et al. Volumetric, nanoscale optical imaging of mouse and human kidney via expansion microscopy. Sci. Rep. 8, 10396 (2018).

    Article  Google Scholar 

  34. 34.

    Tsai, A. et al. Nuclear microenvironments modulate transcription from low-affinity enhancers. eLife 6, e28975 (2017).

    Article  Google Scholar 

  35. 35.

    Jiang, N. et al. Superresolution imaging of Drosophila tissues using expansion microscopy. Mol. Biol. Cell 29, 1413–1421 (2018).

    CAS  Article  Google Scholar 

  36. 36.

    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 

  37. 37.

    Sümbül, U. et al. Automated scalable segmentation of neurons from multispectral images. In Advances in Neural Information Processing Systems 29 (eds Lee, D. D. et al.) 1912–1920 (NIPS Foundation, La Jolla, CA, 2016).

  38. 38.

    Mosca, T. J., Luginbuhl, D. J., Wang, I. E. & Luo, L. Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons. eLife 6, 1–29 (2017).

    Article  Google Scholar 

  39. 39.

    Wang, I. E., Lapan, S. W., Scimone, M. L., Clandinin, T. R. & Reddien, P. W. Hedgehog signaling regulates gene expression in planarian glia. eLife 5, e16996 (2016).

    Article  Google Scholar 

  40. 40.

    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 

  41. 41.

    Artur, C. G. et al. Plasmonic nanoparticle-based expansion microscopy with surface-enhanced Raman and dark-field spectroscopic imaging. Biomed. Opt. Express 9, 603–615 (2018).

    Article  Google Scholar 

  42. 42.

    Villaseñor, R., Schilling, M., Sundaresan, J., Lutz, Y. & Collin, L. Sorting tubules regulate blood-brain barrier transcytosis. Cell Rep. 21, 3256–3270 (2017).

    Article  Google Scholar 

  43. 43.

    Li, R., Chen, X., Lin, Z., Wang, Y. & Sun, Y. Expansion enhanced nanoscopy. Nanoscale 10, 17552–17556 (2018).

    CAS  Article  Google Scholar 

  44. 44.

    Treweek, J. B. et al. Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nat. Protoc. 10, 1860–1896 (2015).

    CAS  Article  Google Scholar 

  45. 45.

    Unnersjö-Jess, D. et al. Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion. Kidney Int. 93, 1008–1013 (2018).

    Article  Google Scholar 

  46. 46.

    Wei, L. et al. Super-multiplex vibrational imaging. Nature 544, 465–470 (2017).

    CAS  Article  Google Scholar 

  47. 47.

    Hu, F. et al. Supermultiplexed optical imaging and barcoding with engineered polyynes. Nat. Methods 15, 194–200 (2018).

    CAS  Article  Google Scholar 

  48. 48.

    Kumar, A. et al. Influenza virus exploits tunneling nanotubes for cell-to-cell spread. Sci. Rep. 7, 40360 (2017).

    CAS  Article  Google Scholar 

  49. 49.

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

    CAS  Article  Google Scholar 

  50. 50.

    Elmore, J. G. et al. Diagnostic concordance among pathologists interpreting breast biopsy specimens. J. Am. Med. Assoc. 313, 1122–1132 (2015).

    CAS  Article  Google Scholar 

  51. 51.

    Choi, H. M. T. et al. Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat. Biotechnol. 28, 1208–1212 (2010).

    CAS  Article  Google Scholar 

  52. 52.

    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  Article  Google Scholar 

  53. 53.

    Lin, R. et al. A hybridization-chain-reaction-based method for amplifying immunosignals. Nat. Methods 15, 275–278 (2018).

    CAS  Article  Google Scholar 

  54. 54.

    Lee, J. H. et al. Highly multiplexed subcellular RNA sequencing in situ. Science 343, 1360–1363 (2014).

    CAS  Article  Google Scholar 

  55. 55.

    Ke, R. et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nat. Methods 10, 857–860 (2013).

    CAS  Article  Google Scholar 

  56. 56.

    Yoon, Y.-G. et al. Feasibility of 3D reconstruction of neural morphology using expansion microscopy and barcode-guided agglomeration. Front. Comput. Neurosci. 11, 97 (2017).

    Article  Google Scholar 

  57. 57.

    Lubeck, E. & Cai, L. Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat. Methods 9, 743–748 (2012).

    CAS  Article  Google Scholar 

  58. 58.

    Lubeck, E., Coskun, A. F., Zhiyentayev, T., Ahmad, M. & Cai, L. Single-cell in situ RNA profiling by sequential hybridization. Nat. Methods 11, 360–361 (2014).

    CAS  Article  Google Scholar 

  59. 59.

    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).

    Article  Google Scholar 

  60. 60.

    Moffitt, J. R. et al. High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization. Proc. Natl Acad. Sci. USA 113, 11046–11051 (2016).

    CAS  Article  Google Scholar 

  61. 61.

    Wang, G., Moffitt, J. R. & Zhuang, X. Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy. Sci. Rep. 8, 4847 (2018).

    Article  Google Scholar 

  62. 62.

    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  Article  Google Scholar 

  63. 63.

    Wang, Y. et al. Rapid sequential in situ multiplexing with DNA-exchange-imaging in neuronal cells and tissues. Nano Lett. 17, 6131–6139 (2017).

    CAS  Article  Google Scholar 

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E.S.B. acknowledges the NIH (1R01NS102727, 1R01EB024261, 1R41MH112318, 1R01MH110932, 1RM1HG008525, and Director's Pioneer Award 1DP1NS087724), the Open Philanthropy Project, DARPA, John Doerr, the NSF (grant 1734870), the MIT Aging Brain Initiative/Ludwig Foundation, the HHMI-Simons Faculty Scholars Program, IARPA D16PC00008, the US Army Research Laboratory and the US Army Research Office under contract/grant number W911NF1510548, the US–Israel Binational Science Foundation (grant 2014509), the MIT Media Lab, the MIT Brain and Cognitive Sciences Department, and the McGovern Institute. A.T.W. acknowledges the Hertz Foundation Fellowship.

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A.T.W., Y.Z., and E.S.B. all contributed to the writing of the manuscript and have read and agreed to its content.

Corresponding author

Correspondence to Edward S. Boyden.

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Competing interests

E.S.B. is a co-inventor on multiple patents related to ExM and is also a co-founder of a company ( commercializing ExM. Y.Z. and A.T.W. are inventors on several inventions related to ExM.

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

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