A practical guide to optimization in X10 expansion microscopy

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Expansion microscopy is a relatively new approach to super-resolution imaging that uses expandable hydrogels to isotropically increase the physical distance between fluorophores in biological samples such as cell cultures or tissue slices. The classic gel recipe results in an expansion factor of ~4×, with a resolution of 60–80 nm. We have recently developed X10 microscopy, which uses a gel that achieves an expansion factor of ~10×, with a resolution of ~25 nm. Here, we provide a step-by-step protocol for X10 expansion microscopy. A typical experiment consists of seven sequential stages: (i) immunostaining, (ii) anchoring, (iii) polymerization, (iv) homogenization, (v) expansion, (vi) imaging, and (vii) validation. The protocol presented here includes recommendations for optimization, pitfalls and their solutions, and detailed guidelines that should increase reproducibility. Although our protocol focuses on X10 expansion microscopy, we detail which of these suggestions are also applicable to classic fourfold expansion microscopy. We exemplify our protocol using primary hippocampal neurons from rats, but our approach can be used with other primary cells or cultured cell lines of interest. This protocol will enable any researcher with basic experience in immunostainings and access to an epifluorescence microscope to perform super-resolution microscopy with X10. The procedure takes 3 d and requires ~5 h of actively handling the sample for labeling and expansion, and another ~3 h for imaging and analysis.

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Fig. 1: Outline and timing of the X10 expansion microscopy protocol.
Fig. 2: Anchoring and homogenization conditions can be optimized for the specific requirements of the sample and experiment.
Fig. 3: Assembly of a gelation chamber (Step 17).
Fig. 4: Removal of the gel from the gelation chamber and assembly of a digestion chamber (Step 22).
Fig. 5: Expansion of the homogenized sample (Steps 24 and 25).
Fig. 6: Assembly of an imaging chamber (Steps 26 and 27).
Fig. 7: Exemplary images of samples before and after expansion, and quantification of expansion factor and distortions.
Fig. 8: Expected results, exemplary images of samples before and after expansion.

Code availability

The code used and described in this paper is available as Supplementary Data 1. Additional advice on how to use it can be obtained from the authors upon reasonable request.

Data availability

All data shown in this paper are available from the authors upon reasonable request.


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We thank E. De Gaulejac and D. Lorenz for critically reading the manuscript. We thank S. Kabatas for helpful discussions. S.T. has received funding, as an ISTplus Fellow, from the European Union’s Horizon 2020 Research and Innovation Programme under Marie Skłodowska-Curie grant agreement no. 754411. J.G.D. gratefully acknowledges funding by the Austrian Science Fund (FWF; I 3600-B27). This work was further supported by grants to S.O.R. from the European Research Council (ERC-2013-CoG NeuroMolAnatomy) and the Deutsche Forschungsgemeinschaft (DFG; SFB1286/Z03).

Author information

S.T., J.G.D., and S.O.R. prepared the manuscript. S.T. prepared the figures. S.T. performed the experiments and imaging. C.S. wrote the Python Anaconda scripts provided here for alignment of pre- and post-expansion images, expansion factor determination, and distortion analysis.

Correspondence to Sven Truckenbrodt.

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Key reference using this protocol

Truckenbrodt, S. et al. EMBO Rep. 19, e45836 (2018): http://embor.embopress.org/content/early/2018/07/09/embr.201845836

Integrated supplementary information

Supplementary Figure 1 Measuring the expansion factor manually.

(a) The same images as shown in Fig. 7 for determining the expansion factor via the script provided with this manuscript are used here for manual determination of the expansion factor. 10 distance measurements were taken, using the respective tool in ImageJ, between landmark points that are easily identifiable in the pre- and post-expansion image. The lines for distance measurements were separately drawn manually in both images. Corresponding lines are numbered in both images. Scale bar: 20 µm. (b) Results of the same 10 manual distance measurements, together with the ratio of the measurement after expansion over the measurement before expansion. The average ratio is indicated below, and serves as the expansion factor for this image: 7.98. The manual measurement is consistent with the automatic measurement via the script (expansion factor: 8.0, see Fig. 7), but is considerably more time-consuming.

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Supplementary Text and Figures

Supplementary Figure 1

Reporting Summary

Supplementary Data 1

Script for analyzing pre- and post-expansion images.

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Truckenbrodt, S., Sommer, C., Rizzoli, S.O. et al. A practical guide to optimization in X10 expansion microscopy. Nat Protoc 14, 832–863 (2019) doi:10.1038/s41596-018-0117-3

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