Kim, D.; Latshaw, A.; Balkota, M.; Wiggert, M.; Alata, M.; Huang, S.; Constant, S.; Maechler, P.; Vanden Berghe, P.; Bonacina, L.

Airway epithelium plays a major role as the primary interface between human body and the external environment, acting both as a physical and functional barrier. In vitro airway models that reproduce the epithelium architecture are therefore a valuable tool for studying infection, inflammation, and transport processes. In this work, we present a label-free, non-invasive method to visualize and measure mucociliary transport in air-liquid human models using third-harmonic generation (THG) microscopy with an optical parametric amplifier laser source at 1300 nm. By exploiting the intrinsic nonlinear contrast at optical heterogeneities, THG provides high resolution images of both epithelial structures and of the overlying mucus layer without the need for fluorescence staining or sample processing. Time-lapse THG imaging reveals depth-dependent transport dynamics within the mucus, offering new insights into mucociliary transport mechanism. Our approach offers a physiologically relevant way to assess mucociliary function in vitro and could support studies on respiratory diseases, drug delivery and efficacy, and epithelial remodeling.