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Photo-expansion microscopy enables super-resolution imaging of cells embedded in 3D hydrogels

Abstract

Hydrogels are extensively used as tunable, biomimetic three-dimensional cell culture matrices, but optically deep, high-resolution images are often difficult to obtain, limiting nanoscale quantification of cell–matrix interactions and outside-in signalling. Here we present photopolymerized hydrogels for expansion microscopy that enable optical clearance and tunable ×4.6–6.7 homogeneous expansion of not only monolayer cell cultures and tissue sections, but cells embedded within hydrogels. The photopolymerized hydrogels for expansion microscopy formulation relies on a rapid photoinitiated thiol/acrylate mixed-mode polymerization that is not inhibited by oxygen and decouples monomer diffusion from polymerization, which is particularly beneficial when expanding cells embedded within hydrogels. Using this technology, we visualize human mesenchymal stem cells and their interactions with nascently deposited proteins at <120 nm resolution when cultured in proteolytically degradable synthetic polyethylene glycol hydrogels. Results support the notion that focal adhesion maturation requires cellular fibronectin deposition; nuclear deformation precedes cellular spreading; and human mesenchymal stem cells display cell-surface metalloproteinases for matrix remodelling.

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Fig. 1: PhotoExM process and formulations.
Fig. 2: Isotropy and resolution of PhotoExM process.
Fig. 3: Super-resolution imaging of hMSCs and nascent deposited matrix in synthetic hydrogels.
Fig. 4: Nuclear architecture of hMSCs and evidence for cell-surface metalloproteinases.

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Data availability

The data used to prepare the figures, all displayed microscopy images and the raw images used to prepare these images are available at https://doi.org/10.25810/2eb5-8x48.

Code availability

The codes used for B-spline registration of the pre- and post-expansion images displayed in Fig. 2a are freely available at Koon et al.52.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (DE016523 and DK120921 to K.S.A. and AR049446 to B.B.O.). We thank J. Dragavon and the BioFrontiers Institute Advanced Light Microscopy Core (RRID, SCR 018302) for the discussions and support with confocal microscopes used in this study: a Nikon A1R confocal microscope via a National Institute of Standards and Technology University of Colorado (CU) cooperative grant (70NANB15H226) and an Imaris Workstation funded by a National Institutes of Health grant (1S10RR026680-01A1).

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K.A.G., B.B.O., E.S.B. and K.S.A. designed the experiments. K.A.G., T.-L.C., N.P.S. and V.V.R. carried out GtG experiments. K.A.G., L.J.M., N.P.S. and T.E.B. designed and prepared the PhotoExM formulations. T.-L.C., A.A.C. and J.S.S. carried out the expansion of muscle tissue sections and myofibres. K.A.G., N.P.S., C.Z. and C.-C.Y. carried out the non-rigid registration experiments. N.P.S. carried out the fluorophore photobleaching experiments. K.A.G. and K.S.A. wrote the paper. All authors contributed to the discussion of the data.

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Correspondence to Kristi S. Anseth.

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E.S.B. discloses cofounding a company that pursues commercial applications of expansion microscopy. B.B.O. discloses a potential conflict of interest as a Scientific Advisory Board Member for Satellos Biosciences. The other authors declare no competing interests.

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Günay, K.A., Chang, TL., Skillin, N.P. et al. Photo-expansion microscopy enables super-resolution imaging of cells embedded in 3D hydrogels. Nat. Mater. 22, 777–785 (2023). https://doi.org/10.1038/s41563-023-01558-5

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