Welcome to the website for expansion microscopy protocols.
Here you can find relevant papers, protocols, materials lists, and advice on expansion microscopy (ExM), a new technology for imaging biological specimens with fine detail by physically making them bigger through a chemical process that preserves nanoscale isotropy. It enables super-resolution imaging on a conventional light microscope.
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) (*, 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]
Reviews and tutorials
Gao R.*, Asano S.M.*, Boyden E.S. (2017) Q&A: Expansion microscopy, BMC Biology
15:50. (*, co-first authors) [Link to paper]
Application papers from our group
Freifeld L, Odstrcil I, Förster 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
, doi:10.1073/pnas.1706281114. [Epub ahead of print]
[Link to paper]
Talk at TED.com, "A new way to study the brain's invisible secrets," filmed June 2016. [Link]
Please send inquiries to
Expansion microscopy (ExM) enables large 3-D volumes to be imaged with nanoscale precision, even on ordinary microscopes. It works by physically magnifying the specimen directly, in contrast to traditional optical magnification methods. The protocols below show you how to label your specimens, anchor key labels or biomolecules to the swellable polymer that you will form evenly throughout specimens, mechanically homogenize samples, and expand the resultant tissue-material composites.
All ExM protocols obey a standard workflow -- anchor key biomolecules or labels to an evenly synthesized, swellable polymer, mechanically homogenize the specimen, and then expand (followed by perhaps further labeling and amplification steps). This can be applied to human clinical specimens (ExPath, Nature Biotechnology 2017), to the visualization of RNA (ExFISH, Nature Methods 2016), and to the visualization of proteins (including genetically encoded fluorophores and antibodies) (proExM, Nature Biotechnology 2016). The kind of label can also be engineered for specific purposes - for example, the original DNA oligonucleotide-anchoring form of ExM (ExM 1.0, Science 2015), used to make the initial discovery of isotropic biological specimen expansion, turns out to greatly faciltiate an iterative protocol that expands a single sample over and over for added resolution (iExM, Nature Methods 2017) - see the "Explore" page for links to the primary papers.
Detailed protocols are found below. Please contact us at firstname.lastname@example.org with questions or suggestions.
ExM 1.0 Materials List, v1.3
This materials list contains an inventory of all the materials needed to carry out the ExM 1.0 protocol (Science
ExPath protocol for human clinical specimens, V1.1
A detailed description of how to expand human clinical specimens, using the expansion pathology (ExPath) protocol (Nature Biotechnology
Iterative expansion microscopy, V1.0
A detailed description of how to expand cells or tissues twice, using the iterative expansion (iExM) protocol (Nature Methods
ExFISH Protocol For Cells and Tissues, V1.0
A detailed description of how to expand cells or tissues according to the ExFISH protocol (Nature Methods
proExM Protocol For Tissues, V1.0
A detailed description of how to expand tissues according to the proExM protocol (Nature Biotechnology
proExM Protocol For Cultured Cells, V1.0
A detailed description of how to expand cultured cells according to the proExM protocol (Nature Biotechnology
ExM 1.0 Cultured Cell Protocol, V1.4
A detailed description of how to expand cultured cells according to the ExM 1.0 protocol (Science
ExM 1.0 Brain Slice Protocol, V1.4
A detailed description of how to expand brain slices according to the ExM 1.0 protocol (Science
ExM 1.0 DNA-Antibody Conjugation Protocol, V1.2
How to create ExM 1.0-ready secondary antibodies for the ExM 1.0 protocol (Science
proExM for tissues: gelation demonstration
A videorecording of the gelation and digestion steps relevant to proExM, ExFISH, and other ExM protocols, when applied to tissues.
The protocols here posted, and the videos, should be enough for most people familiar with immunohistochemistry to get going. At times we have conducted hands-on workshops such as the Janelia Research Campus expansion microscopy workshop (2016), as well as two workshops at UCSD/Salk Institute (2016, 2017). We also host people regularly to visit our group at MIT to see how we do expansion microscopy. For more info on this training program, click here