Third harmonic generation microscopy reveals structure and mucus dynamics in human airway epithelium models

Airway epithelium is the primary interface between human body and the external environment, acting as a physical and functional barrier. In vitro airway models that reproduce the epithelium architecture are valuable tools 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 1,300 nm. By exploiting the intrinsic nonlinear contrast at optical heterogeneities, THG provides high-resolution images of both epithelial structures and the overlying mucus layer without fluorescence staining or sample processing as recently shown for other lung-tissue environments. Time-lapse THG imaging reveals depth-dependent transport dynamics within the mucus, offering new insights into mucociliary transport. Our approach offers a physiologically relevant way to assess mucociliary function in vitro and could support studies on respiratory diseases, drug delivery and epithelial remodeling.