Microscale imaging of cilia-driven fluid flow

Abstract

Cilia-driven fluid flow is important for multiple processes in the body, including respiratory mucus clearance, gamete transport in the oviduct, right-left patterning in the embryonic node, and cerebrospinal fluid circulation. Multiple imaging techniques have been applied toward quantifying ciliary flow. Here, we review common velocimetry methods of quantifying fluid flow. We then discuss four important optical modalities, including light microscopy, epifluorescence, confocal microscopy, and optical coherence tomography, that have been used to investigate cilia-driven flow.

Fig. 1

Anatomic sites of ciliary action. Cilia are found in several organ systems, including (left) the respiratory and nasal tract, where the airway surface liquid is divided into the periciliary layer and the mucus layer, (middle) the ventricles of the brain and the oviduct, where no such layered structure exists, and (right) the transient embryonic node, in which ciliary-driven flow is thought to be generated by rotating cilia

Fig. 2

Ciliary physiology spans many length scales, requiring multiple imaging modalities (top). Ciliary ultrastructure such as microtubules and motor proteins can be imaged by transmission electron microscopy (TEM) (image reproduced from [3]) (middle). Microfluidic flow can be imaged by optical modalities such as light microscopy, epifluorescence, confocal, and optical coherence tomography (OCT). The image here shows cilia in the ventricle as visualized by confocal fluorescence (image reproduced from [157]) (bottom). Anatomic clearance of mucus can be quantified by radiotracer methods (image reproduced from [187]) and computed X-ray tomography (CT)

Fig. 3

Workflow of ciliary imaging. For any given choice of flow system (left column) to be imaged, an optical imaging modality can be chosen (middle column) and combined with a choice of velocimetry technique (right column). Typically, the imaging modality will determine contrast and cross-sectional ability, while the velocimetry technique will determine the extent of velocimetry information recovered. CSF cerebrospinal fluid. OCT optical coherence tomography. PTV particle tracking velocimetry. DPIV digital particle image velocimetry

Fig. 4

Flow field generated by a single nodal cilium in a zebrafish embryo, reproduced from [169]. Here, the flow field specifies a magnitude (color) and direction of the velocity vector at every location in space, relative to epithelium (axes denoted as d–v dorsal ventral, a–p anterior posterior, and l–r left right). The flow field was estimated by employing three-dimensional, confocal fluorescent particle tracking velocimetry

Fig. 5

Commonly used optical techniques for investigating cilia-driven fluid flow, with advantages and disadvantages of each technique. Top Light microscopy has historically been used (image showing tracheal mucus velocity measurement, reproduced from [108]). Middle Epifluorescence (image showing epithelial ciliary flow, unpublished work from authors) and confocal fluorescence (image showing airway surface liquid, reproduced from [165]) are the current standard methods. Bottom Optical coherence tomography (image showing epithelial ciliary flow, unpublished work from authors) is an emerging method showing promise for flow quantification

Fig. 6

Early works in the videomicroscopic study of ciliary physiology. Panels (a) and (b) are two consecutive frames from a 200 frames-per-second movie of abfrontal cilia motion in Mytilus edulis (blue mussel). Images reproduced from [96], published in 1930. Panels (c) and (d) are closely spaced frames from a movie that captured cilia-driven fluid flow in a rabbit trachea. Red blood cells were used as flow tracers. Images are reproduced from [188], originally produced in 1934. The scale bar in (b) is for frames (a) and (b) only

Fig. 7

Quantification of ciliary performance using light microscopy and entropy of dye mixing, reproduced from [115]. (a) Microfluidic chip layout; (b, c) time evolution of mixing process, false color insets show contrast-enhanced flow patterns; (d) time-varying image entropy, a measure of flow performance; (e) maps of local entropy, another measure of flow performance

Fig. 8

Micro-optical coherence tomography (µOCT) image showing airway surface liquid, reproduced from [183]. OCT can be used to differentiate the mucus layer (mu), periciliary layer (pcl), and individual cilia (green arrows) in relation to epithelial cell layer (ep)