Abstract
A straightforward ex vivo approach has been developed and refined to enable high-resolution imaging and quantitative assessment of motile cilia function in mouse airway epithelial tissue, allowing critical insights into cilia motility and cilia generated flow using different mouse models or following different sample treatments. In this method, freshly excised mouse trachea is cut longitudinally through the trachealis muscle which is then sandwiched between glass coverslips within a thin silicon gasket. By orienting the tissue along its longitudinal axis, the natural curling of the trachealis muscle helps maintain the sample in a configuration optimal for imaging along the full tracheal length. High-speed video microscopy, utilizing differential interference contrast (DIC) optics and a fast digital camera capturing at >200 frames per second is then used to record ciliary motion. This enables detailed measurement of both cilia beat frequency (CBF) and waveform characteristics. The application of 1 µm microspheres to the bathing media during imaging allows for additional analysis of fluid flow generated by ciliary activity. The entire procedure typically takes around 40 min to complete per animal: ~30 min for tissue harvest and sample mounting, then ~10 min for imaging samples and acquiring data.