The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy of RNA. We developed a small-molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, postexpansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity as well as single-molecule precision in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) separates RNAs and supports amplification of single-molecule signals (i.e., via hybridization chain reaction) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine.

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Lightsheet imaging was performed in the W.M. Keck Facility for Biological Imaging at the Whitehead Institute for Biomedical Research. We would like to acknowledge W. Salmon for assistance with the Zeiss Z.1 lightsheet, S. Olenych from Carl Zeiss Microscopy for providing the microscopy filters, and H.T. Choi and N. Pierce for advice and consultation on HCR. E.R.D. is supported by NIH CEGS grant P50 HG005550, NIH CEGS grant 1 RM1 HG008525, and NSF GRF grant DGE1144152. A.T.W. acknowledges the Hertz Foundation Fellowship. F.C. acknowledges the NSF Fellowship and Poitras Fellowship. AR and AC acknowledge support from NIH/NHLBI grant 1U01HL129998. E.S.B. acknowledges support by the New York Stem Cell Foundation–Robertson Award, NSF CBET 1053233, MIT Media Lab Consortium, the MIT Synthetic Intelligence Project, NIH Director's Pioneer Award 1DP1NS087724, NIH 2R01DA029639, NIH Director's Transformative Award 1R01MH103910, NIH 1R24MH106075, IARPA D16PC00008, the Open Philanthropy Project, and Jeremy and Joyce Wertheimer. J.-B.C. was supported by a Simons Postdoctoral Fellowship.

Author information

Author notes

    • Fei Chen
    •  & Asmamaw T Wassie

    These authors contributed equally to this work.


  1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Fei Chen
    • , Asmamaw T Wassie
    •  & Edward S Boyden
  2. Media Lab, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Fei Chen
    • , Asmamaw T Wassie
    • , Shahar Alon
    • , Shoh Asano
    • , Jae-Byum Chang
    • , Adam Marblestone
    •  & Edward S Boyden
  3. McGovern Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Fei Chen
    • , Asmamaw T Wassie
    • , Shahar Alon
    • , Shoh Asano
    • , Jae-Byum Chang
    • , Adam Marblestone
    •  & Edward S Boyden
  4. Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Allison J Cote
    •  & Arjun Raj
  5. Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Anubhav Sinha
  6. Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA.

    • Evan R Daugharthy
    •  & George M Church
  7. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Evan R Daugharthy
  8. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • George M Church
  9. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Edward S Boyden


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F.C., A.T.W., E.R.D., A.M., G.M.C., and E.S.B. conceived RNA-tethering strategies to the ExM gel. F.C. and A.T.W. conceived and developed the LabelX reagent. F.C., A.T.W., J.-B.C., and S. Alon developed ExM gel stabilization by re-embedding. F.C., A.T.W., and E.R.D. conceived and developed reversible HCR strategies. F.C., A.T.W., and E.S.B. designed, and F.C. and A.T.W. performed experiments. A.J.C. and A.R. provided FISH reagents and guidance on usage, and A.J.C. performed experiments. A.S. performed data analysis. S. Asano performed lightsheet imaging and analysis. E.S.B. supervised the project. F.C., A.T.W., A.S., and E.S.B. wrote the paper, and all authors contributed edits and revisions.

Competing interests

F.C., A.T.W., S. Alon, E.R.D., J.-B.C., A.M., G.M.C., and E.S.B. are inventors on one or more patents or patent applications related to the technologies here discussed. E.S.B. is cofounder of Expansion Technologies, a company whose goal is to facilitate access to expansion microscopy technologies for the scientific community.

Corresponding author

Correspondence to Edward S Boyden.

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    Supplementary Text and Figures

    Supplementary Figures 1–12 and Supplementary Tables 1–3

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    Supplementary Table 4

    RNA FISH Probe Sequences


  1. 1.

    Volume rendering of lightsheet microscopy of ExFISH

    Volume rendering of Thy1-YFP (green) brain tissue acquired by lightsheet microscopy with HCR-ExFISH targeting YFP (red) and Gad1 (blue) mRNA.

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