Welcome to the website for expansion microscopy protocols.

Here you can find relevant papers and protocols for expansion microscopy (ExM), a powerful technology for imaging biological specimens with fine detail by physically making them bigger through an isotropic chemical expansion process that preserves nanoscale detail. It enables nanoscale resolution imaging on a conventional light microscope, and decrowds biomolecules from one another for better chemical analysis.

Perhaps thousands of groups have begun to use expansion microscopy to probe human cells, brain circuits, cancer biopsies, microbes, tissue specimens of all kinds (including from human patients), and other kinds of biological specimen; over 700 papers and preprints performing expansion microscopy experiments have appeared to date.

This website contains papers and protocols from the Synthetic Neurobiology lab (Boyden lab) at MIT. These protocols are of general use and have been applied to a great many species, tissues, and/or cell types. In addition to these protocols, many groups have begun to make their own customized or specialized forms of expansion microscopy, and we encourage you to look for papers and protocols appropriate for the species, tissue, and/or cell type of interest for your scientific question (e.g., you could start by adding your species, tissue, and/or cell type of interest to this Google Scholar search).

Papers

Technology papers

Alice E. Stanton*, Jinyoung Kang*, Joel W. Blanchard, Carles A. Boix, Margaret E. Schroeder, Youngmi Lee, Hanquan Su, Shiwei Wang, Eunah Yu, Amauche Emenari, Zhuyu Peng, Emre Agbas, Oyku Cerit, Demian Park, Ruihan Zhang, David A. Bennett, Peng Yin, Manolis Kellis, Robert Langer, Edward Boyden**, Li-Huei Tsai** (2026) Expansion Revealing of Pathology Resolves Nanostructures Associated with Inflammatory Phenotypes in COVID-19 Decedent Human Brain Tissue, bioRxiv 2026.05.14.725177; doi: https://doi.org/10.64898/2026.05.14.725177. (*, contributed equally; **, co-corresponding) [Link to paper]

Helena Hu, Donatus Krah, Antonios Ntolkeras, Sushovan Chanda, Alina Heimbrodt, Milton Mondal, Jonas Altendorf, Bowen Jing, Bonnie Berger, Ali H. Shaib*, Silvio Rizzoli*, Edward S Boyden* (2026) Thousandfold Expansion Microscopy, bioRxiv 2026.05.31.729018; doi: https://doi.org/10.64898/2026.05.31.729018. (*, co-corresponding) [Link to paper]

Shin TW, Wang H*, Zhang C*, An B, Lu Y, Zhang E, Lu X, Karagiannis ED, Kang JS, Emenari A, Symvoulidis P, Asano S, Lin L, Costa EK; IMAXT Grand Challenge Consortium; Marblestone AH, Kasthuri N, Tsai LH, Boyden ES (2025) Dense, continuous membrane labeling and expansion microscopy visualization of ultrastructure in tissues, Nature Communications 16(1):1579. (*, contributed equally) [Link to paper]

Kang J*, Schroeder ME*, Lee Y, Kapoor C, Yu E, Tarr TB, Titterton K, Zeng M, Park D, Niederst E, Wei D, Feng G, Boyden ES (2024) Multiplexed expansion revealing for imaging multiprotein nanostructures in healthy and diseased brain, Nature Communications 15(1):9722. (*, contributed equally) [Link to paper]

Wang S*, Shin TW*, Yoder HB 2nd, McMillan RB, Su H, Liu Y, Zhang C, Leung KS, Yin P, Kiessling LL**, Boyden ES** (2024) Single-shot 20-fold expansion microscopy, Nature Methods, doi:10.1038/s41592-024-02454-9. Online ahead of print. (*, equal contribution; **, co-corresponding authors) [Link to paper]

Valdes PA, Yu CJ*, Aronson J*, Ghosh D, Zhao Y, An B, Bernstock JD, Bhere D, Felicella MM, Viapiano MS, Shah K, Chiocca EA**, Boyden ES** (2024) Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding, Science Translational Medicine 16(732):eabo0049. (*, contributed equally; **, contributed equally) [Link to paper]

Cui Y*, Yang G*, Goodwin DR, O'Flanagan CH, Sinha A, Zhang C, Kitko KE, Shin TW, Park D, Aparicio S; CRUK IMAXT Grand Challenge Consortium; Boyden ES (2023) Expansion microscopy using a single anchor molecule for high-yield multiplexed imaging of proteins and RNAs, PLoS One 18(9):e0291506. (*, contributed equally) [Link to paper]

Deblina Sarkar*, Jinyoung Kang*, Asmamaw T. Wassie*, Margaret E. Schroeder, Zhuyu Peng, Tyler B. Tarr, Ai-Hui Tang, Emily D. Niederst, Jennie Z. Young, Hanquan Su, Demian Park, Peng Yin, Li-Huei Tsai**, Thomas A. Blanpied** & Edward S. Boyden** (2022) Revealing nanostructures in brain tissue via protein decrowding by iterative expansion microscopy, Nature Biomedical Engineering 6:1057-1073. (*, contributed equally; **, co-corresponding) [Link to paper]

Gao R*, Yu CJ*, Gao L, Piatkevich KD, Neve RL, Munro JB, Upadhyayula S, Boyden ES (2021) A highly homogeneous polymer composed of tetrahedron-like monomers for high-isotropy expansion microscopy, Nature Nanotechnology 16:698-707. (*, equal contribution) [Link to paper]

Alon S*, Goodwin DR*, Sinha A*, Wassie AT*, Chen F*, Daugharthy ER**, Bando Y, Kajita A, Xue AG, Marrett K, Prior R, Cui Y, Payne AC, Yao CC, Suk HJ, Wang R, Yu CJ, Tillberg P, Reginato P, Pak N, Liu S, Punthambaker S, Iyer EPR, Kohman RE, Miller JA, Lein ES, Lako A, Cullen N, Rodig S, Helvie K, Abravanel DL, Wagle N, Johnson BE, Klughammer J, Slyper M, Waldman J, Jane-Valbuena J, Rozenblatt-Rosen O, Regev A; IMAXT Consortium, Church GM***+, Marblestone AH***, Boyden ES***+ (2021) Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems, Science 371(6528):eaax2656. (* equal contribution, ** key contributions to early stages of project, *** equal contribution, +co-corresponding authors) [Link to paper]

Yu CJ, Barry NC, Wassie AT, Sinha A, Bhattacharya A, Asano S, Zhang C, Chen F, Hobert O, Goodman MB, Haspel G, Boyden ES (2020) Expansion microscopy of C. elegans, eLife 9:e46249. [Link to paper]

Emmanouil D. Karagiannis*, Jeong Seuk Kang*, Tay Won Shin, Amauche Emenari, Shoh Asano, Leanne Lin, Emma K. Costa, IMAXT Grand Challenge Consortium, Adam H. Marblestone, Narayanan Kasthuri, Edward S. Boyden (2019) Expansion Microscopy of Lipid Membranes, bioRxiv 829903. (*, equal contribution) [Link to paper]

Ruixuan Gao*, Shoh M. Asano*, Srigokul Upadhyayula*, Igor Pisarev, Daniel E. Milkie, Tsung-Li Liu, Ved Singh, Austin Graves, Grace H. Huynh, Yongxin Zhao, John Bogovic, Jennifer Colonell, Carolyn M. Ott, Christopher Zugates, Susan Tappan, Alfredo Rodriguez, Kishore R. Mosaliganti, Shu-Hsien Sheu, H. Amalia Pasolli, Song Pang, C. Shan Xu, Sean G. Megason, Harald Hess, Jennifer Lippincott-Schwartz, Adam Hantman, Gerald M. Rubin, Tom Kirchhausen, Stephan Saalfeld, Yoshinori Aso, Edward S. Boyden**, Eric Betzig** (2019) Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution, Science 363(6424):eaau8302. (*, equal contribution, **, co-corresponding) [Link to paper]

Freifeld L, Odstrcil I, Forster D, Ramirez A, Gagnon JA, Randlett O, Costa EK, Asano S, Celiker OT, Gao R, Martin-Alarcon DA, Reginato P, Dick C, Chen L, Schoppik D, Engert F, Baier H, Boyden ES (2017) Expansion microscopy of zebrafish for neuroscience and developmental biology studies, Proceedings of the National Academy of Sciences 114(50):E10799-E10808. [Link to paper]

Zhao Y*, Bucur O*, Irshad H, Chen F, Weins A, Stancu AL, Oh EY, DiStasio M, Torous V, Glass B, Stillman IE, Schnitt SJ, Beck AH**, Boyden ES** (2017) Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy, Nature Biotechnology 35(8):757-764. (*, co-first authors; **, co-corresponding authors) [Link to paper] [Link to code]

Chang, J.-B., Chen, F., Yoon, Y.-G., Jung, E. E., Babcock H., Kang J.-S., Asano S., Suk H.-J., Pak N., Tillberg P.W., Wassie A., Cai D., Boyden E.S. (2017) Iterative expansion microscopy, Nature Methods 14:593-599. [Link to paper]

Chen, F.*, Wassie, A.T.*, Cote, A.J., Sinha, A., Alon, S., Asano, S., Daugharthy, E.R., Chang, J.-B., Marblestone, A., Church, G.M., Raj, A., Boyden, E.S. (2016) Nanoscale Imaging of RNA with Expansion Microscopy, Nature Methods 13(8):679-84. (*, co-first authors) [Link to paper]

Tillberg, P.W.*, Chen, F.*, Piatkevich, K.D., Zhao, Y., Yu, C.-C., English, B.P., Gao, L., Martorell, A., Suk, H.-J., Yoshida, F., DeGennaro, E.M., Roossien, D.H., Gong, G., Seneviratne, U., Tannenbaum, S.R., Desimone, R., Cai, D., Boyden, E.S. (2016) Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies, Nature Biotechnology 34:987-992. (*, co-first authors) [Link to paper]

Chen, F.*, Tillberg, P.W.*, Boyden, E.S. (2015) Expansion Microscopy, Science 347(6221):543-548. (*, equal contribution) [Link to paper]

Protocols and hands-on-tutorial papers

Yu CJ*, Orozco Cosio DM*, Boyden ES (2022) ExCel: Super-Resolution Imaging of C. elegans with Expansion Microscopy, Methods in Molecular Biology 2468:141-203. (* contributed equally) [Link to paper]

Zhang C*, Kang JS*, Asano SM, Gao R, Boyden ES (2020) Expansion Microscopy for Beginners: Visualizing Microtubules in Expanded Cultured HeLa Cells, Current Protocols in Neuroscience 92(1):e96. (*, contributed equally) [Link to paper]

Bucur O*, Fu F*, Calderon M, Mylvaganam GH, Ly NL, Day J, Watkin S, Walker BD, Boyden ES**, Zhao Y** (2020) Nanoscale imaging of clinical specimens using conventional and rapid expansion pathology, Nature Protocols 15:1649-1672. (*, equal contribution, **, co-corresponding) [Link to paper]

Asano SM*, Gao R*, Wassie AT*, Tillberg PW, Chen F, Boyden ES (2018) Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissues, Current Protocols in Cell Biology 80(1):e56. (*, co-first authors) [Link to paper]

Reviews and perspective pieces

Ha V. Vo*, Rong Xu*, Edward S. Boyden** & Yongxin Zhao** (2026) Expansion Microscopy, Nature Reviews Methods Primers 6:28 (2026). (*, contributed equally; **, co-corresponding) [Link to paper]

Wassie AT*, Zhao Y*, Boyden ES (2019) Expansion microscopy: principles and uses in biological research, Nature Methods 16(1):33-41. (*, equal contributors) [Link to paper]

Boyden, E. S. (2018) Physical magnification of objects, Materials Horizons 6:11-13. [Link to paper]

Alon, S.*, Huynh, G. H.*, Boyden, E. S. (2018) Expansion Microscopy: Enabling Single Cell Analysis In Intact Biological Systems, The FEBS Journal 286(8):1482-1494. (*, co-first authors) [Link to paper]

Karagiannis ED, Boyden ES (2018) Expansion microscopy: development and neuroscience applications, Current Opinion in Neurobiology 50:56-63. [Link to paper]

Gao R.*, Asano S.M.*, Boyden E.S. (2017) Q&A: Expansion microscopy, BMC Biology 15:50. (*, co-first authors) [Link to paper]

Videos

Talk at TED.com, "A new way to study the brain's invisible secrets," filmed June 2016. [Link]

Contact Information

Please send inquiries to info@expansionmicroscopy.org

Current protocols

20ExM (protocol for 20-fold expansion in a single step), V1.0 [Link to PDF for download (see Supp Note 1, p. 35 of the PDF)]
A detailed description of how to expand cells and tissues using the 20ExM single-shot 20x expansion protocol. (Nature Methods 2024).

dExPath (decrowding expansion pathology, with post-expansion staining) protocol for human clinical specimens, V1.0 [Link to PDF for download]
A detailed description of how to expand human clinical specimens, for post-expansion antibody visualization of proteins, using the decrowding expansion pathology (dExPath) protocol (Science Translational Medicine 2024).

proExM tutorial exercise, using cultured cell lines [Link to website and PDF for download]
A tutorial-like lab protocol for beginners to learn expansion microscopy (the proExM variant, for protein visualization), using a cultured cell line and an antibody against a specific protein. Appropriate for workshops and classrooms.

ExCel protocol for expanding C. elegans, V5.0 [Link to website and PDF for download]
A detailed description of how to expand C. elegans, for antibody visualization of proteins and in situ hybridization visualization of RNAs, using the expansion of C. elegans (ExCel) protocols (three versions: standard, epitope-preserving, and iterative; eLife 2020). Version 4.0 is posted [here] for historical reasons, but we recommend you start with Version 5.0, above.

ExPath (expansion pathology, with pre-expansion staining) protocol for human clinical specimens, V2.0 [Link to website and PDF for download]
A detailed description of how to expand human clinical specimens, for antibody visualization of proteins, using the expansion pathology (ExPath) protocol (Nature Biotechnology 2017). Version 1.1 is posted [here] for historical reasons, but we recommend you start with Version 2.0, above.

Iterative expansion microscopy, V1.0 [PDF]
A detailed description of how to expand cells or tissues twice, using the iterative expansion (iExM) protocol, for antibody visualization of proteins (Nature Methods 2017).

ExFISH protocol for cells and tissues, V1.1 [Link to website and PDF for download]
A detailed description of how to expand cells or tissues according to the ExFISH protocol, for in situ hybridization visualization of RNAs (Nature Methods 2016). Version 1.0 is posted here for historical reasons, but we recommend you start with Version 1.1, above.

proExM protocol for tissues, V1.1 [Link to website and PDF for download]
A detailed description of how to expand tissues according to the proExM protocol, for antibody visualization of proteins (Nature Biotechnology 2016). Version 1.0 is posted here for historical reasons, but we recommend you start with Version 1.1, above.

proExM protocol for cultured cells, V1.1 [Link to website and PDF for download]
A detailed description of how to expand cultured cells according to the proExM protocol, for antibody visualization of proteins (Nature Biotechnology 2016). Version 1.0 is posted here for historical reasons, but we recommend you start with Version 1.1, above.

Older protocols

ExM 1.0 cultured cell protocol, V1.4 [Word]
A detailed description of how to expand antibody-stained cultured cells according to the ExM 1.0 protocol (Science 2015).
This materials list [Excel format] contains an inventory of all the materials needed to carry out the ExM 1.0 protocol (Science 2015). This protocol is presented for historical purposes, but if you are just starting out, please consider the newer "proExM Protocol For Cultured Cells" above, which is more current and simpler.

ExM 1.0 brain slice protocol, V1.4 [Word]
A detailed description of how to expand antibody-stained brain slices according to the ExM 1.0 protocol (Science 2015).
This materials list [Excel format] contains an inventory of all the materials needed to carry out the ExM 1.0 protocol (Science 2015). This protocol is presented for historical purposes, but if you are just starting out, please consider the newer "proExM Protocol For Tissues" above, which is more current and simpler.

ExM 1.0 DNA-Antibody Conjugation Protocol, V1.2 [Word]
How to create ExM 1.0-ready secondary antibodies for the ExM 1.0 protocol (Science 2015).
This materials list [Excel format] contains an inventory of all the materials needed to carry out the ExM 1.0 protocol (Science 2015). This protocol is presented for historical purposes, but if you are just starting out, please consider the newer proExM protocols above, which are more current and simpler.

Hardware

3D-Printable Sample Holder for Zeiss Z.1 Lightsheet Microscope V1.0 [Description (Word)] [3-d printable file]
Description, and downloadable 3-d printer file, for our 3D-printable expansion microscopy sample holder for the Zeiss Z.1 Lightsheet Microscope. As described in Current Protocols in Cell Biology 2018.

Videos

proExM for tissues: gelation demonstration [Youtube video]
A videorecording of the gelation and digestion steps relevant to proExM, ExFISH, and other ExM protocols, when applied to tissues.

Classes

The protocols here posted, and the videos, should be enough for most people familiar with basic immunohistochemistry and microscopy to get going on expansion microscopy. At times we have conducted hands-on workshops at various universities and institutes, as well as at summer schools and short courses. There are also online message boards and other resources to provide community around the topic of expansion microscopy.