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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Chen, F., Tillberg, P.W. & Boyden, E.S. Optical imaging. Expansion microscopy. Science 347, 543–548 (2015).
Femino, A.M., Fay, F.S., Fogarty, K. & Singer, R.H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).
Levsky, J.M. & Singer, R.H. Fluorescence in situ hybridization: past, present and future. J. Cell Sci. 116, 2833–2838 (2003).
Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).
Choi, H.M.T. et al. Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat. Biotechnol. 28, 1208–1212 (2010).
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).
Cajigas, I.J. et al. The local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging. Neuron 74, 453–466 (2012).
Wang, F. et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. 14, 22–29 (2012).
Tillberg, P.W. et al. Expansion microscopy of biological specimens with protein retention. Nat. Biotechnol. doi:10.1038/nbt.3625 (2016).
Chozinski, T.J. et al. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat. Methods 13, 485–488 (2016).
Engreitz, J.M. et al. The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341, 1237973 (2013).
Panning, B., Dausman, J. & Jaenisch, R. X chromosome inactivation is mediated by Xist RNA stabilization. Cell 90, 907–916 (1997).
Plath, K., Mlynarczyk-Evans, S., Nusinow, D.A. & Panning, B. Xist RNA and the mechanism of X chromosome inactivation. Annu. Rev. Genet. 36, 233–278 (2002).
Mito, M., Kawaguchi, T., Hirose, T. & Nakagawa, S. Simultaneous multicolor detection of RNA and proteins using super-resolution microscopy. Methods 98, 158–165 (2015).
Clemson, C.M. et al. An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol. Cell 33, 717–726 (2009).
Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009).
Lubeck, E. & Cai, L. Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat. Methods 9, 743–748 (2012).
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).
Chen, K.H., Boettiger, A.N., Moffitt, J.R., Wang, S. & Zhuang, X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).
Beliveau, B.J. et al. Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc. Natl. Acad. Sci. USA 109, 21301–21306 (2012).
Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).
Lein, E.S. et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176 (2007).
Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H.K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).
Batish, M., van den Bogaard, P., Kramer, F.R. & Tyagi, S. Neuronal mRNAs travel singly into dendrites. Proc. Natl. Acad. Sci. USA 109, 4645–4650 (2012).
Cabili, M.N. et al. Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution. Genome Biol. 16, 20 (2015).
Zhang, D.Y. & Seelig, G. Dynamic DNA nanotechnology using strand-displacement reactions. Nat. Chem. 3, 103–113 (2011).
Lee, J.H. et al. Highly multiplexed subcellular RNA sequencing in situ. Science 343, 1360–1363 (2014).
Ke, R. et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nat. Methods 10, 857–860 (2013).
Shah, S. et al. Single-molecule RNA detection at depth via hybridization chain reaction and tissue hydrogel embedding and clearing. Development (in the press).
Bruchez, M. et al. Semiconductor nanocrystals as fluorescent biological labels. Science 281, 2013–2016 (1998).
Fouz, M.F. et al. Bright fluorescent nanotags from bottlebrush polymers with DNA-tipped bristles. ACS Cent. Sci. 1, 431–438 (2015).
Steward, O., Wallace, C.S., Lyford, G.L. & Worley, P.F. Synaptic activation causes the mRNA for the IEG Arc to localize selectively near activated postsynaptic sites on dendrites. Neuron 21, 741–751 (1998).
Buckley, P.T. et al. Cytoplasmic intron sequence-retaining transcripts can be dendritically targeted via ID element retrotransposons. Neuron 69, 877–884 (2011).
Steward, O. & Schuman, E.M. Compartmentalized synthesis and degradation of proteins in neurons. Neuron 40, 347–359 (2003).
Buxbaum, A.R., Wu, B. & Singer, R.H. Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability. Science 343, 419–422 (2014).
Jung, H., Yoon, B.C. & Holt, C.E. Axonal mRNA localization and local protein synthesis in nervous system assembly, maintenance and repair. Nat. Rev. Neurosci. 13, 308–324 (2012).
Raj, A. & Tyagi, S. Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Methods Enzymol. 472, 365–386 (2010).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Thévenaz, P., Ruttimann, U.E. & Unser, M. A pyramid approach to subpixel registration based on intensity. IEEE Trans. Image Process. 7, 27–41 (1998).
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.
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.
Integrated supplementary information
(a) Epi-fluorescence image of single molecule FISH (smFISH) against GAPDH on HeLa cells expanded without LabelX treatment. (b) Epi-fluorescence image of smFISH performed against GAPDH on expanded HeLa cells treated with LabelX. Images are maximum intensity projections of 3-D stacks. Nuclei stained with DAPI (shown in blue). Scale bars: 20 μm (post-expanded units).
To access the effect of LabelX on fluorescent in situ hybridization, fixed HeLa cells were stained with smFISH probe-sets, followed by DNAse I treatment to remove the staining. The cells were then treated with LabelX and stained again with the same smFISH probe-sets. (a) UBC staining before LabelX treatment and (b) UBC staining after probe removal and LabelX treatment. (c) EEF2 staining before LabelX treatment. (d) EEF2 staining after probe removal and LabelX treatment. (e) Comparison of smFISH spots counted for individual cells before LabelX, and after probe removal and application of LabelX. The number of RNA molecules detected in a given cell was quantified using an automated spot counting algorithm (n=7 cells for each bar). Plotted are mean + standard error; no significant difference in spot counts before vs after LabelX (p > 0.5 for before vs. after for UBC, p > 0.5 for before vs. after for EEF2; t-test, unpaired, two-tailed). Images in a-d are maximum intensity projections of 3-D stacks; scale bars: 10 μm (pre-expanded units).
Different RNA species spanning 3 orders of magnitude in abundance were detected via single molecule RNA fluorescent in situ hybridization (FISH) in HeLa cells before and after ExM with LabelX treatment (shown in Fig. 1e). (a) Ratio of FISH spots detected after expansion to spots detected before expansion for single cells. Representative before vs. after ExFISH images shown: (b,c) TFRC; (d,e) GAPDH; (f,g) ACTB. Scale bars, 10 μm (pre-expanded units) in b, d, f; c, e, g, expanded physical size 21 μm (imaged in PBS).
(a) Pre-expansion widefield image of a cultured HeLa cell stained with DAPI to visualize the nucleus (top panel) and smFISH probes against ACTB (bottom panel). (b) Post-expansion widefield image of the same cell as in (a). (c) Pre-expansion widefield image of LabelX treated Thy1-YFP brain slice (Right panel, YFP protein) stained with DAPI (Left panel) (MIP, 4 μm z-depth). (d) Post-expansion image of the same region as in (c) (MIP, 12 μm). (e) Ratio of the expansion factor of cell bodies for individual cells to the expansion factor of their respective nuclei. smFISH stain is used to outline the boundaries of the cell bodies of cultured cells while the endogenous YFP protein is used to demarcate the cell bodies of neurons in Thy1-YFP brain slices. Plotted are mean ± standard error. The ratio for both cultured cells and brain slices did not significantly deviate from one (p >0.05 for both, 1-sample t-test; n = 6, cultured HeLa cells; n = 7, cells in 1 brain slice). Scale bars, 10 μm.
(a) Representative FISH image of TOP2A in a single HeLa cell before expansion (MIP of cell thickness). (b) ExFISH image of cell in (a) taken with the same optical parameters. (c) Merged image of (a) and (b) (red and green for before and after expansion respectively); distance measurements between pairs of mRNA spots before (L, red line) and after (L’, green line; note that these lines overlap nearly completely) expansion were used to quantify expansion isotropy. (d) Mean of the absolute value of the measurement error (i.e., |L-L’|) plotted against measurement length (L) for all pairs of mRNA spots (mean ± standard deviation, N = 4 samples, 6.8 x 105 measurements). Scale bars: white, 10 µm pre-expansion units; blue, white scale bar divided by expansion factor. Orange line indicates diffraction limit of the microscope used (see Methods for details).
(a) Five consecutive widefield fluorescence images (top to bottom, then left to right) of GAPDH, applied to the cell of Fig. 2a. (b) Widefield fluorescence images showing ExFISH with serially delivered probes against six RNA targets (right to left, then top to bottom: NEAT1, EEF2, ACTB, UBC, GAPDH, and USF2) in a cultured HeLa cell (raw images of composite shown in Fig. 2e). Scale bars: 20 μm in expanded units.
FISH probes bearing HCR initiators are hybridized to a target mRNA. During amplification, metastable DNA hairpins bearing fluorophores assemble into polymer chains onto the initiators, thus amplifying signal downstream of the FISH probe hybridization event.
(a) Widefield image of a LabelX treated Thy1-YFP brain slice (YFP protein, green) stained with probes against YFP (red) and Gad1 (magenta) followed by HCR amplification. Probes against YFP transcripts were amplified with the B1 amplifier set (see Methods) while probes against Gad1 transcripts were amplified with the B2 amplifier set (MIP, 59 μm). (b) Widefield image of LabelX treated Thy1-YFP brain slice (YFP protein, green) treated with the same HCR amplifiers as in (a) (namely B1 (red) and B2 (magenta)) without the addition of probes (MIP, 50 μm). (c) HCR spots detected per volume of expanded sample. Analysis was performed on samples which were either treated or not treated with FISH probes followed by HCR amplification. An automated spot counting algorithm (as used in Fig. 1) was used to count HCR spots. The endogenous YFP protein was used to delineate regions used for the analysis. Plotted are mean ± standard error. HCR spot counts are significantly different in the presence of probes than without probes (p <0.05 for both B1 and B2 amplifier sets, Welch’s t-test; n=4 fields of view each). Scale bars: 50 μm.
(a) Volume rendering of Thy1-YFP (green) brain tissue acquired by lightsheet microscopy with HCR-ExFISH targeting YFP (red) and Gad1 (blue) mRNA. (b) A maximum intensity projection (~8 µm in Z) of a small subsection of the volume, showing the high resolution of imaging and single molecule localization of imaging expanded specimens with lightsheet imaging (scale bar: 10 µm, in pre-expansion units, expansion factor, 3×). (c) Zoom in of the volume rendering in (a) (scale bar: 20 µm, in pre-expansion units, 3×).
Supplementary Figure 10 Two-color co-localization of FISH probes with HCR amplification in expanded Thy1-YFP brain slices.
(a) Schematic showing two color amplification of the same target. A transcript of interest is targeted by probes against alternating parts of the sequence, and bearing two different HCR initiators, allowing for amplification in two colors. (b) Confocal image showing FISH staining with HCR amplification against the Camk2a transcript in two colors (red and blue; YFP fluorescence shown in green). (c) The result of an automated two-color spot co-localization analysis performed on the data set shown in (b). Each purple spot represents a positive co-localization identified by the algorithm and overlaid on the confocal image of YFP. Zoom in of dendrites showing two color FISH staining with HCR amplification against Camk2a (d,e) and Dlg4 (f,g) transcripts. Top row shows the raw two color staining data corresponding to the bottom row showing co-localized spots identified by the automated algorithm (replicated from Fig. 3j-k for convenience). Scale bars: (b,c) 10 μm (3×); (d-g) 2 μm (3×). (b-g) are MIP of ~1.6 μm thickness in unexpanded coordinates.
(a) Schematic for HCR amplification and reversal. HCR amplification is initiated with custom-made HCR hairpins bearing toe-holds for toe-hold mediated strand displacement. After amplification, the addition of a disassembling strand initiates the disassembly of the HCR polymers via strand displacement. (b) ExFISH-treated Thy1-YFP brain slice (YFP in blue) is shown stained with YFP FISH probes bearing HCR initiators and amplified with custom made HCR hairpins bearing toe-holds for strand displacement (green dots). The different panels show the state of HCR reversal at different times after the addition of strands to initiate the disassembly of the HCR polymers. Scale bars: 20 μm (in post-expansion units).
Supplementary Figure 12 Dependence of RNA FISH spot intensity on degree of expansion and concentration of LabelX.
HeLa cells, treated with LabelX diluted to different final concentrations of Label-IT Amine concentration, were expanded and stained with a probe-set against GAPDH. After staining, the gelled samples were expanded in 1× PBS (~2× expansion ratio) and water (~4× expansion ratio) and the spot intensity for the different samples was quantified. Plotted are mean + standard error; N = 6 cells.
Supplementary Figures 1–12 and Supplementary Tables 1–3 (PDF 2151 kb)
RNA FISH Probe Sequences (PDF 576 kb)
Volume rendering of Thy1-YFP (green) brain tissue acquired by lightsheet microscopy with HCR-ExFISH targeting YFP (red) and Gad1 (blue) mRNA. (MP4 50373 kb)
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Chen, F., Wassie, A., Cote, A. et al. Nanoscale imaging of RNA with expansion microscopy. Nat Methods 13, 679–684 (2016). https://doi.org/10.1038/nmeth.3899
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