Multiscale imaging of polarity in bovine oviductal organoids

Organoids are three-dimensional cell culture systems that recapitulate key structural features of their tissue of origin; however, interpretation of organoid architecture is inherently dependent on the imaging modality used. In organoids cultured within extracellular matrix (ECM), cells typically adopt an apical-in/basal-out configuration in which the apical surface faces an enclosed lumen, limiting direct visualization and access to the luminal surface. Experimental manipulation of polarity enables the generation of defined structural stages that can be leveraged to evaluate how imaging modalities resolve epithelial organization. In this study, ECM-embedded (apical-in) and suspension-cultured after ECM removal (apical-out) bovine oviductal organoids were used as controlled structural models for comparing imaging techniques across spatial scales. Apical-out organoids were generated by ECM removal and centrifugation, exposing the luminal epithelial surface. Using a multiscale imaging approach-including brightfield and time-lapse microscopy, confocal immunofluorescence with line-scan analysis, total internal reflection fluorescence microscopy (TIRF), and electron microscopy-we assessed how each modality captures distinct features of the epithelial surface. Brightfield imaging enabled rapid assessment of overall morphology but did not resolve membrane orientation, while time-lapse imaging revealed differences in organoid dynamics and surface behavior. Confocal microscopy demonstrated redistribution of cortical F-actin and overall epithelial organization within intact structures, whereas TIRF microscopy provided surface-restricted visualization consistent with external accessibility of actin-rich surface structures. Electron microscopy further resolved ultrastructural features, including microvillar projections, supporting differences in membrane-surface orientation at nanometer resolution. Across modalities, consistent structural differences between apical-in and apical-out organoids highlight how polarity-defined states influence the interpretation of organoid architecture. Collectively, this study presents a multiscale imaging framework for characterizing epithelial organization in organoid systems and demonstrates how polarity-defined structural states can be leveraged to improve interpretation across imaging modalities. These findings provide a foundation for standardized structural characterization of organoids and support their application in reproductive biology, including studies of epithelial-embryo interactions and luminal signaling.