ABSTRACT
Mucociliary clearance, characterized by mucus secretion and its conveyance by ciliary action, is a fundamental physiological process that plays an important role in host defense. Although it is known that ciliary activity changes with chemical and mechanical stimuli, the autoregulatory mechanisms that govern ciliary activity and mucus transport in response to normal and pathophysiological variations in mucus are not clear. We have developed a high-speed, 1-µm-resolution, cross-sectional imaging modality, termed micro-optical coherence tomography (µOCT), which provides the first integrated view of the functional microanatomy of the epithelial surface. We monitored invasion of the periciliary liquid (PCL) layer by mucus in fully differentiated human bronchial epithelial cultures and full thickness swine trachea using µOCT. We further monitored mucociliary transport (MCT) and intracellular calcium concentration simultaneously during invasion of the PCL layer by mucus using colocalized µOCT and confocal fluorescence microscopy in cell cultures. Ciliary beating and mucus transport are up-regulated via a calcium-dependent pathway when mucus causes a reduction in the PCL layer and cilia height. When the load exceeds a physiological limit of approximately 2 µm, this gravity-independent autoregulatory mechanism can no longer compensate, resulting in diminished ciliary motion and abrogation of stimulated MCT. A fundamental integrated mechanism with specific operating limits governs MCT in the lung and fails when periciliary layer compression and mucus viscosity exceeds normal physiologic limits.
Subject(s)
Epithelial Cells/metabolism , Mucociliary Clearance , Mucus/metabolism , Respiratory Mucosa/metabolism , Trachea/metabolism , Animals , Calcium/metabolism , Cells, Cultured , Cilia/metabolism , Homeostasis , Humans , Microscopy, Confocal , Microscopy, Fluorescence , Time Factors , Tomography, Optical Coherence/methods , ViscosityABSTRACT
Active illumination microscopy (AIM) is a method of redistributing dynamic range in a scanning microscope using real-time feedback to control illumination power on a sub-pixel time scale. We describe and demonstrate a fully integrated instrument that performs both feedback and image reconstruction. The image is reconstructed on a logarithmic scale to accommodate the dynamic range benefits of AIM in a single output channel. A theoretical and computational analysis of the influence of noise on active illumination feedback is presented, along with imaging examples illustrating the benefits of AIM. While AIM is applicable to any type of scanning microscope, we apply it here specifically to two-photon microscopy.
