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Click-ExM enables expansion microscopy for all biomolecules

Nature Methods volume 18, pages 107–113 (2021)Cite this article

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Abstract

Expansion microscopy (ExM) allows super-resolution imaging on conventional fluorescence microscopes, but has been limited to proteins and nucleic acids. Here we develop click-ExM, which integrates click labeling into ExM to enable a ‘one-stop-shop’ method for nanoscale imaging of various types of biomolecule. By click labeling with biotin and staining with fluorescently labeled streptavidin, a large range of biomolecules can be imaged by the standard ExM procedure normally used for proteins. Using 18 clickable labels, we demonstrate click-ExM on lipids, glycans, proteins, DNA, RNA and small molecules. We demonstrate that click-ExM is applicable in cell culture systems and for tissue imaging. We further show that click-ExM is compatible with signal-amplification techniques and two-color imaging. Click-ExM thus provides a convenient and versatile method for super-resolution imaging, which may be routinely used for cell and tissue samples.

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Fig. 1: Schematic of the click-ExM workflow.
Fig. 2: Click-ExM imaging of lipids.
Fig. 3: Click-ExM imaging of glycans.
Fig. 4: Click-ExM imaging of proteins, nucleic acids and small molecules.

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Data availability

Source data for Figs. 2c,e,g,j and 3d,g,h and Extended Data Figs. 1c,e,f,i,j, 3f, 5c, 6h,k, 7e, 8c,h and 9c,f,j are available with this paper. All other data in this study are available from the corresponding author upon reasonable request.

Code availability

The MATLAB codes and examples for registration and distortion analysis are available from GitHub (https://github.com/Yujie-S/Click-ExM_data_process_and_example).

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Acknowledgements

We thank S. Wang (Peking University) and Y. Rao (Peking University) for assistance on cell and tissue sample preparation, W. Zhou at the Analytical Instrumentation Center of Peking University for help on matrix-assisted laser desorption/ionization mass spectrometry, Y. Liang (Carl Zeiss, Shanghai Co., Ltd) for help on Elyra Lattice structured illumination microscopy and LSM 900 Airyscan imaging and L. Gu for helpful discussions. This work is supported by the National Key Research and Development Projects (grant no. 2018YFA0507600 to X.C.) and the National Natural Science Foundation of China (nos. 91753206 and 21521003 to X.C.).

Author information

Author notes

  1. Yujie Shi

    Present address: Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA

  2. Yuntao Zhu

    Present address: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

Authors and Affiliations

  1. College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    De-en Sun, Xinqi Fan, Yujie Shi, Hao Zhang, Zhimin Huang, Bo Cheng, Qi Tang, Wei Li, Yuntao Zhu, Jinyi Bai, Xiaoguang Lei & Xing Chen

  2. Beijing National Laboratory for Molecular Sciences, Peking University, Beijing, China

    De-en Sun, Xinqi Fan, Yujie Shi, Hao Zhang, Bo Cheng, Qi Tang, Wei Li, Yuntao Zhu, Xiaoguang Lei & Xing Chen

  3. Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Zhimin Huang, Jinyi Bai, Xiaoguang Lei & Xing Chen

  4. School of Life Sciences, Peking University, Beijing, China

    Wei Liu, Yuan Li & Xiaoting Wang

  5. Synthetic and Functional Biomolecules Center, Peking University, Beijing, China

    Xiaoguang Lei & Xing Chen

  6. Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China

    Xiaoguang Lei & Xing Chen

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Contributions

X.C. conceived the study and supervised the project. X.C. and D.S. designed the experiments and analyzed the data. D.S. conducted most of the experiments unless specified otherwise. X.F., H.Z., B.C., Q.T., W.L. and Y.Z. performed most of the chemical synthesis. J.B. synthesized Az-afatinib under the supervision of X.L. Y.S. and D.S. performed expansion factor calculation and distortion analysis. Z.H., W.L., Y.L. and X.W. prepared primary cell and tissue samples. D.S. and X.C. wrote the manuscript with input from all the authors.

Corresponding author

Correspondence to Xing Chen.

Ethics declarations

Competing interests

A Chinese patent application (application no. 201911150397.1) covering the use of click-ExM has been filed in which the Peking University is the applicant, and X.C. and D.S. are the inventors.

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Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Adaption of the protein-retention ExM (proExM) protocol for click-ExM.

HeLa cells were immunostained for α-tubulin. a, Post-proExM image of α-tubulin (AF555, red hot, top left) partially overlaid with the corresponding pre-expansion image (bottom right). AcX was used for anchoring. sEF, expansion factor determined by transformation matrix after rigid registration. b, Zoomed-in view of the boxed region in a showing corresponding pre-proExM (left) and post-proExM (right) images of α-tubulin. c, Fluorescence intensity profiles along the lines in b. a.u., arbitrary units. d-f, Diameter of α-tubulin resolved from the zoomed-in view of the boxed region in a, showing the post-proExM image (d), a representative cross-sectional intensity profile of α-tubulin (dots) with Gaussian fitting (solid line) (e), and distribution of Gaussian-fitted full-width-at-half-maximum (FWHM) of the intensity profiles along yellow lines in d, yielding an average FWHM of 83 ± 7 nm (mean ± s.d., n = 170) (f). g-i, Nanoscale isotropy of proExM. Pre- and post-ExM images (g) were aligned using rigid registration. Post-ExM images before and after non-rigid registration were overlaid and shown in green and magenta, respectively (h). The yellow arrows in h indicate the distortion vector field. From the boxed region in h, the root mean square (RMS) error was quantified as a function of measured distance (i), in which the black line indicates mean value and the grey area indicates ± s.d. j, Quantification of gEF, expansion factor determined by the gel size. From 161 gels of both proExM and click-ExM experiments, an average gEF was determined to be 4.5 ± 0.1 (mean ± s.d.). Scale bars: 5 μm (a, d, g, h) and 2 μm (b). All distances and scale bars correspond to the pre-expansion dimensions based on sEF. Representative images from ≥3 independent experiments.

Source data

Extended Data Fig. 2 Click-ExM preserves lipid labeling signal by streptavidin-fluorophore conjugates.

a, The cells were treated with Alk-Cho to metabolically label Cho-containing phospholipids, followed by reaction with azide-dye and the standard proExM procedure. b, Pre- and post-ExM images of COS-7 cells treated with Alk-Cho and reacted with azide-TAMRA (red hot). c, Pre- and post-ExM images of COS-7 cells stained with the lipophilic dye, DiI (red hot). d, Click-ExM images of COS-7 cells treated with Alk-Cho, reacted with azide-biotin, and stained with SA-AF555 (red hot) was compatible with anchoring with AcX (top) and GA (bottom). The nuclei were stained by Hoechst 33342 (cyan). AcX (b, c) was used for anchoring. Scale bars: 5 μm (b-d). All distances and scale bars correspond to the pre-expansion dimensions based on gEF. Representative images from ≥3 (d) or 2 (b, c) independent experiments.

Extended Data Fig. 3 Pre- and post-click-ExM imaging of lipids (Related to Fig. 2).

a, Pre- and post-click-ExM images of Alk-Cho-labeled phospholipids (AF555, magenta) in COS-7 cells. Mito-GFP (green) was expressed in the mitochondrial matrix and Tom20 located in the mitochondrial outer membrane was immunostained with AF647 (red). b, Pre- and post-click-ExM images of Alk-Cho-labeled phospholipids (AF555, magenta) and ER expressing Sec61β-GFP (green) in COS-7 cells. c, Pre- and post-click-ExM images of Alk-Cho-labeled phospholipids (AF555, magenta) and Golgi expressing b4Gal-T1-GFP (green) in COS-7 cells. The same post-click-ExM images were shown in Fig. 2. d-f, Nanoscale isotropy of click-ExM for imaging of Alk-Cho-labeled phospholipids. Pre- and post-click-ExM images (d, AF488, red hot) were aligned using rigid registration. Post-click-ExM images before and after non-rigid registration were overlaid and shown in green and magenta, respectively (e). The yellow arrows in e indicate the distortion vector field. From the boxed region in e, the RMS error was quantified as a function of measured distance (f), in which the black line indicates mean value and the grey area indicates ± s.d. g, Pre- and post-click-ExM images of Alk-Cho-labeled phospholipids (AF555, cyan hot) in rat cardiomyocytes. (h) Zoomed-in view of the boxed region from the pre-click-ExM image in g. i, Zoomed-in view of the boxed region from the post-click-ExM image in g. j, Pre- and post-click-ExM images of lipids in cells metabolically labeled with various bioorthgonal reporters including Alk-palmitic acid, Alk-farnesol, Alk-myristic acid and Alk-stearic acid (AF555, cyan hot). COS-7 cells were used for Alk-farnesol labeling and HeLa cells were used for other lipids labeling. k, Pre- and post-click-ExM images of 4-hydroxy-2-nonenal (HNE)-labeled protein carbonylation (AF555, cyan hot) in HeLa cells. Cysteine residues were reacted with HNE through Michael addition, reacted with aminooxy-alkyne (AOyne), and followed with the click-ExM workflow. GA (a-c, g-i) or AcX (d, e, j, k) was used for anchoring. Scale bars: 10 μm (a-e, g, j, k) and 2 μm (h, i). All distances and scale bars correspond to the pre-expansion dimensions based on gEF (a-c, g-k) or sEF (d-f). Analysis of isotropy from ≥3 (d-f) independent experiments. Representative images from ≥3 (g-i, pre-click-ExM images of j) or 2 (a-c, k, post-click-ExM images of j) independent experiments.

Source data

Extended Data Fig. 4 Click-ExM imaging of Cho-containing phospholipids in the mouse brain.

a–c, Acute mouse brain slices with a thickness of 150 µm were metabolically labeled with Alk-Cho. Post-click-ExM image with an imaging depth of ~23 μm (a) and 3D rendering images of post-click-ExM (b, c) are shown. Cho-containing phospholipids were stained with SA-AF555 in a and b or with SA-AF488 in c. Images were acquired using a 20×/0.75-NA air objective for a or a 40×/1.10-NA water objective for b and c. The nuclei were stained with Hoechst 33342 (cyan) in b and c. Scale bars: 20 μm. All distances and scale bars correspond to the pre-expansion dimensions based on sEF. Representative images from ≥3 slices.

Extended Data Fig. 5 Signal amplification by immunosignal hybridization chain reaction (isHCR) or by a biotin trimer.

a, Workflow of isHCR for signal amplification. Cells click-labeled with streptavidin-dye were incubated with DNA-biotin HCR initiators, and then incubated with a pair of DNA-fluorophore HCR amplifiers for signal amplification. b, Workflow of iterative signal amplification by using the biotin trimer. c, Fluorescence imaging of HeLa cells with α-tubulin immunostained with AF555, which was amplified by using the biotin trimer. Procedures of iterative signal amplification (top). HeLa cells were immunostained for α-tubulin, and five labeling conditions were used for comparison from left to right: (i) conventional antibody staining with AF555-conjugated secondary antibody, (ii) biotin-conjugated secondary antibody and streptavidin-AF555, (iii)-(v) one, two and four rounds of iterative staining by biotin trimer and streptavidin-AF555. Representative images of each conditions were shown (middle). Histogram shows mean fluorescence intensity quantification in five labeling conditions (n = 5 fields of view per sample, bottom left). Bars represent the mean value and error bars represent the s.d. The P values are listed, and P < 0.05 was considered significant. One-way ANOVA with post hoc Tukey HSD calculator was used for statistical analysis. Magnified image for four rounds signal amplification of immunostained α-tubulin in HeLa cells was shown (bottom right). Scale bars: 50 μm (c, middle) and 10 μm (c, bottom right). Representative images from 2 independent experiments.

Source data

Extended Data Fig. 6 Pre- and post-click-ExM imaging of glycans (Related to Fig. 3).

a, Chemical structure of SiaNAz. b, Click-ExM images of sialoglycans (AF555, red hot) in SiaNAz-treated HeLa cells after deconvolution. c, Confocal fluorescence images of rat cardiomyocytes, in which sialoglycans were metabolically labeled with ManNAz (AF555, magenta) and the T-tubule network was immunostained by using the T-tubule marker caveolin-3 (AF488, green). d, Click-ExM images of GalNAz-labeled HeLa cells (AF555, gray) shown in 3D (left) and xy, xz, yz views (right). e, Pre- and post-click-ExM images showing the colocalization between GalNAz-labeled glycans (AF488, green) and immunostained Nup88 (AF546, magenta) in CHO cells. f-h, Nanoscale isotropy of click-ExM for imaging of ManNAz-labeled sialoglycans. Pre- and post-click-ExM images (f, AF488, red hot) were aligned using rigid registration. Post-click-ExM images before and after non-rigid registration were overlaid and shown in green and magenta, respectively (g). The yellow arrows in g indicate the distortion vector field. From the boxed region in g, the RMS error was quantified as a function of measured distance (h), in which the black line indicates mean value and the grey area indicates ± s.d. i-k, Nanoscale isotropy of click-ExM for imaging of GalNAz-labeled glycans. Pre- and post-click-ExM images (i, AF555, red hot) were aligned using rigid registration. Post-click-ExM images before and after non-rigid registration were overlaid and shown in green and magenta, respectively (j). The yellow arrows in j indicate the distortion vector field. From the boxed region in j, the RMS error was quantified as a function of measured distance (k), in which the black line indicates mean value and the grey area indicates ± s.d. l, Pre- and post-click-ExM images of O-GlcNAc distribution chemoenzymatically labeled by Y289L GalT1 in CHO cells (AF555, red hot). m, Pre- and post-click-ExM images of LacNAc-containing glycans (AF488, red hot), which were chemoenzymatically labeled by α1,3-FucT in HeLa cells. Signal was amplified by iterative biotin trimer-streptavidin staining. Scale bars: 20 μm (c), 5 μm (b, d-g, i, j, l, m) and 1 μm (inset of b). All distances and scale bars correspond to the pre-expansion dimensions based on gEF (b, m) or sEF (d-l). AcX was used for anchoring. Analysis of isotropy from 2 (f-k) independent experiments. Representative images from ≥3 (d) or 2 (b, c, e, l, m) independent experiments.

Source data

Extended Data Fig. 7 Pre- and post-click-ExM imaging of proteins (Related to Fig. 4a,b).

a, Pre- and post-click-ExM images of nascent proteins in AHA-treated rat cortical neurons (Fire look-up table, AF488). b, Pre- and post-click-ExM images of HeLa cells treated with AHA, followed by direct click-labeling with alkyne-AFDye 488 (green). The nuclei were stained with Hoechst 33342 (cyan). c-e, Nanoscale isotropy of click-ExM for imaging of AHA-labeled proteins in U2OS cells. Pre- and post-click-ExM images (c, AF555, red hot) were aligned using rigid registration. Post-click-ExM images before and after non-rigid registration were overlaid and shown in green and magenta, respectively (d). The yellow arrows in d indicate the distortion vector field. From the boxed region in d, the RMS error was quantified as a function of measured distance (e), in which the black line indicates mean value and the grey area indicates ± s.d. f, Schematic of click-ExM on specific proteins labeled using the genetic code expansion strategy. The cells were co-transfected with the plasmid encoding a protein of interest bearing the amber codon (POI-TAG) and the plasmid encoding PenK-MbPylRS-tRNApyl CUA in the presence of the Pyl analogue (PenK), followed by the click-ExM procedure. g, Pre- and post-click-ExM images of GFP (AF555, magenta, HEK293T), GFP-histone H2B (AF555, magenta, COS-7) and GTPase KRas (AF488, green, HEK293T). For GFP and GFP-histone H2B, GFP channels were also shown (green). The nuclei were stained with Hoechst 33342 (cyan). Scale bars: 10 μm (a-d) and 5 μm (g). All distances and scale bars correspond to the pre-expansion dimensions based on gEF (a, b, g) or sEF (c-e). AcX was used for anchoring. Analysis of isotropy from 1 (c-e) independent experiment. Representative images from ≥3 (a, pre-click-ExM of g), 2 (post-click-ExM images of GFP in g) or 1 (b, post-click-ExM images of histone H2B and GTPase KRas in g) independent experiments.

Source data

Extended Data Fig. 8 Pre- and post-click-ExM imaging of EdU-labeled nascent DNA and EU-labeled nascent RNA (Related to Fig. 4c,d).

a-c, Nanoscale isotropy of click-ExM for imaging of EdU-labeled DNA. Pre- and post-click-ExM images (a, AF488, red hot) were aligned using rigid registration. Post-click-ExM images before and after non-rigid registration were overlaid and shown in green and magenta, respectively (b). The yellow arrows in b indicate the distortion vector field. From the boxed region in b, the RMS error was quantified as a function of measured distance (c), in which the black line indicates mean value and the grey area indicates ± s.d. The same images were shown in Fig. 4c. d, In U2OS cells, pre- and post-click-ExM images of EdU-labeled nascent DNA (AF555, red hot) in early and middle/late S-phase, from which chromatins with different sizes and shapes were observed. The nuclei were stained with Hoechst 33342 (cyan). e, Click-ExM images showing colocalization of EdU-labeled nascent DNA (AF555, magenta) with immunostained histone H3 (AF488, green). The nuclei were stained with Hoechst 33342 (blue). f-h, Nanoscale isotropy of click-ExM for imaging of EU-labeled RNA. Pre- and post-click-ExM images (f, AF555, red hot) were aligned using rigid registration. Post-click-ExM images before and after non-rigid registration were overlaid and shown in green and magenta, respectively (g). The yellow arrows in g indicate the distortion vector field. From the boxed region in g, the RMS error was quantified as a function of measured distance (h), in which the black line indicates mean value and the grey area indicates ± s.d. The same regions were shown in Fig. 4d. Scale bars: 5 μm. All distances and scale bars correspond to the pre-expansion dimensions based on sEF. AcX was used for anchoring. Analysis of isotropy from 2 (a-c, f-h) independent experiments. Representative images from ≥3 (d), 2 (pre-click-ExM images of e) or 1 (post-click-ExM images of e) independent experiments.

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Extended Data Fig. 9 Pre- and post-click-ExM imaging of small molecules (Related to Fig. 4e-g).

a-c, Nanoscale isotropy of click-ExM for imaging of HeLa cells treated with Az-afatinib. Pre- and post-click-ExM images (a, AF555, red hot) were aligned using rigid registration. Post-click-ExM images before and after non-rigid registration were overlaid and shown in green and magenta, respectively (b). The yellow arrows in b indicate the distortion vector field. From the boxed region in b, the RMS error was quantified as a function of measured distance (c), in which the black line indicates mean value and the grey area indicates ± s.d. The same images were shown in Fig. 4e. (d-f) Nanoscale isotropy of click-ExM for imaging of HeLa cells treated with OP-Puro. Pre- and post-click-ExM images (d, AF488, red hot) were aligned using rigid registration. Post-click-ExM images before and after non-rigid registration were overlaid and shown in green and magenta, respectively (e). The yellow arrows in e indicate the distortion vector field. From the boxed region in e, the RMS error was quantified as a function of measured distance (f), in which the black line indicates mean value and the grey area indicates ± s.d. The same regions were shown in Fig. 4f. g, HeLa cells were labeled with Alk-Hoechst at varied concentrations (0.5–20 μM, AF488, green). The same cells were imaged before and after expansion for comparison except the 0.5 μM group. The same images of the 10 μM group were shown in Fig. 4g. Representative images from 2 independent experiments except 20 μΜ group (1 independent experiment). h-j, Nanoscale isotropy of click-ExM for imaging of HeLa cells labeled with Alk-Hoechst. Pre- and post-click-ExM images (h, AF488, red hot) were aligned using rigid registration. Post-click-ExM images before and after non-rigid registration were overlaid and shown in green and magenta, respectively (i). The yellow arrows in i indicate the distortion vector field. From the boxed region in i, the RMS error was quantified as a function of measured distance (j), in which the black line indicates mean value and the grey area indicates ± s.d. Scale bars: 5 µm. AcX was used for anchoring. All distances and scale bars correspond to the pre-expansion dimensions based on sEF and gEF (0.5 μΜ group in g). Analysis of isotropy from 2 (d-f, h-j) or 1 (a-c) independent experiments.

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Extended Data Fig. 10 Multicolor click-ExM imaging of different biomolecules.

a, Two-color click-ExM imaging of lipids and proteins. COS-7 cells were simultaneously treated with Alk-Cho and AHA. The alkyne-incorporated lipids were reacted with azide-biotin and stained with SA-AF488 (green). The azide-incorporated proteins were reacted with alkyne-TAMRA (magenta). b, Two-color click-ExM imaging of lipids and glycans. COS-7 cells were simultaneously treated with ManNAz and Alk-Cho. The alkyne-incorporated lipids were reacted with azide-biotin and stained with SA-AF555 (magenta). The azide-incorporated glycans were reacted with alkyne-DIG and immunostained with DyLight 488-conjugated anti-DIG antibody (green). c, Two-color click-ExM imaging of glycans and DNA. HeLa cells were simultaneously treated with GalNAz and EdU. The azide-incorporated glycans was reacted with alkyne-biotin and stained with SA-AF488 (green). The alkyne-incorporated nascent DNA was reacted with azide-FLAG and immunostained with anti-FLAG antibody (AF555, magenta). AcX was used for anchoring. Scale bars: 10 μm. All scale bars correspond to the pre-expansion dimensions based on gEF (b) or sEF (a, c). Representative images from ≥3 (pre-click-ExM images of a, c), 2 (pre-click-ExM images of b and post-click-ExM images of a) or 1 (post-click-ExM images of b, c) independent experiments.

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Sun, De., Fan, X., Shi, Y. et al. Click-ExM enables expansion microscopy for all biomolecules. Nat Methods 18, 107–113 (2021). https://doi.org/10.1038/s41592-020-01005-2

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