In Situ Super-Resolution Imaging of Organoids and Extracellular Matrix Interactions via Photoexpansion Microscopy

<b>Introduction/Motivation</b>: Organoids have emerged as the current state-of-the-art multicellular <i>in vitro</i> model for nearly all mammalian tissues and organs. As organoid culture methods have been refined, their utility has become unmatched in developmental biology and disease modelling, owing to their biomimetic cellular composition and 3D architecture, as well as their capacity to match tissue and organ-level functionality on the benchtop. Concurrent to advances in organoid culture, significant progress has been made in 3D imaging, which has enabled characterization of cell composition and fate with spatiotemporal precision. Traditionally, organoids have been imaged by embedding and sectioning or by removal from their culture microenvironment, but the intricacies of the 3D architecture, as well as the role of extracellular matrix (ECM), are lost with these methods. Noninvasive optical sectioning microscopy, such as confocal microscopy, has been used for <i>in situ</i> imaging, but signal attenuation is a persistent issue with confocal imaging techniques, thus requiring additional processing through optical clearing to achieve artifact-free imaging. Additionally, biomolecules smaller than the diffraction limit of light cannot be resolved. To address these numerous limitations, we have developed a method, termed photo-expansion microscopy (PhotoExM), to image organoids grown in natural and synthetic hydrogels. This method imparts a multi-factorial advancement in organoid imaging by overcoming signal attenuation, achieving nanoscale resolution, and facilitating <i>in situ</i> imaging of cell-matrix interactions.<br/><b>Methods</b>: Murine intestinal organoids were cultured in both natural hydrogels (Matrigel) and phototunable PEG hydrogels. Samples were immunostained using standard methods, then fluorophores were linked with acryloyl X to the expansion gel, which was generated by a thiol/acrylate mixed-mode photopolymerization. Unlinked proteins were then digested, the biomaterial was degraded or photo-transferred, and the photopolymerized expansion gel was immersed in diH₂O for expansion. Super-resolution, post-expansion imaging (PostExM) was performed by conventional confocal microscopy. To analyze cell-ECM interactions and ECM dynamics, we developed a custom Matlab script, where we could quantify (on a pixel-by-pixel basis) the thickness of the ECM, as well as integrin-ECM co-localization.<br/><b>Results</b>: Intestinal organoid growth and crypt formation was achieved in both Matrigel and synthetic, phototunable PEG hydrogels. Following fixation and immunolabeling, organoids were isotropically expanded ~4.3x, corresponding to single point resolution of ~120 nm with a conventional confocal microscope. Analysis of PreExM z-stacks revealed striking signal attenuation with ~50% reduction in fluorescent intensity at a depth of just 40 µm, much smaller than the size of many intestinal organoids. PostExM, signal attenuation was eliminated, facilitating imaging of large z-stacks without signal loss. Further, heterogeneities in cell-matrix interactions were imaged and quantified before and after crypt formation, revealing a potential role of differential cell-matrix interactions in organoid symmetry breaking. Finally, using phototunable synthetic hydrogels, we were able to assess the spatial organization of newly synthesized ECM proteins and cell-matrix interactions during organoid growth and crypt formation.<br/><b>Conclusions</b>: Intestinal organoids can be expanded in both Matrigel and synthetic, PEG-based hydrogels to achieve resolution at the nanoscale. The method presented here for PhotoExM also eliminates signal attenuation and can provide broad utility for super-resolution imaging of organoids derived from any tissue, while maintaining 3D architecture and ECM context.<br/><b>Acknowledgements</b>: NIH R01 DK120921, NSF RECODE 2033723