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Motile cilia are cellular organelles that generate directional fluid flow across various epithelial surfaces including the embryonic node and respiratory mucosa. The proper functioning of cilia is necessary for normal embryo development and, for the respiratory system, the clearance of mucus and potentially harmful particulate matter. Here we show that optical coherence tomography (OCT) is well-suited for quantitatively characterizing the microfluidic-scale flow generated by motile cilia. Our imaging focuses on the ciliated epithelium of Xenopus tropicalis embryos, a genetically manipulable and experimentally tractable animal model of human disease. We show qualitative flow profile characterization using OCT-based particle pathline imaging. We show quantitative, two-dimensional, two-component flow velocity field characterization using OCT-based particle tracking velocimetry. Quantitative imaging and phenotyping of cilia-driven fluid flow using OCT will enable more detailed research in ciliary biology and in respiratory medicine.
Fig. 1. Left: Idealized cilia-driven flow seeded with tracer particles (black dots). Velocity vectors v(x,y,z) are open-tipped arrows. The ability of OCT to perform depth-resolved imaging along its depth of focus (DOF) enables visualization of flow generated by a ciliated surface that is largely orthogonal to the optical axis. Right: Scanning electron micrograph of a ciliated Xenopus tropicalis epithelial cell. Each multicilated cell is surrounded by several non-ciliated cells. The scale bar is 5 μm.
Fig. 2. Photomicrograph of stage 36 X. tropicalis embryo in a well for OCT imaging.
Fig. 3. Top: Particle and embryo coordinate system. Bottom: Overview of image processing for OCT-based particle tracking velocimetry. tanâ1 is the four-quadrant arctangent function.
Fig. 4. OCT imaging of X. tropicalis epithelial cilia-driven flow. (a) B-scan of embryo in microparticle-seeded physiologic solution. Original image stack filtered with a 2x2x2 (x,z,t) pixel filter. (b) Background-subtracted B-mode image. The inset in (b) is the minimum projection image across all B-scans over the 5.7 s acquisition. (c) OCT pathline imaging generated by taking the maximum projection over all background-subtracted images over the 5.7 s acquisition. (d) Color-encoding of time pathline imaging. b, body; e, eye; h, head; m, microsphere; t, tail; w, water-air interface. A, anterior; P, posterior. Scale image to have square pixels. Media 1 shows the OCT movie, background-subtracted movie, and related cumulative maximum projection (i.e. cumulative particle pathline) movies.
Fig. 7. OCT-based particle tracking velocimetry of non-recirculatory cilia-driven fluid flow. Note that the same embryo was imaged in Figs. 6 and 7 and only the image well water volume differs between the two image acquisition sessions. (a) shows the particle pathline image and (b) shows the two-dimensional, two-component flow velocity field. The vector arrows at the air/water interface in (b) are artifactual b, body; e, eye; h, head; ps, polystyrene microsphere; t, tail; w, air/water interface. A, anterior; P, posterior.
Fig. 5. Recirculating flow patterns. Each panel is a cumulative maximum projection image (i.e. cumulative particle pathline) through the timestamp on each image. Media 2 is the movie from which these still images were taken.
Fig. 6. OCT-based particle tracking velocimetry of recirculatory/vortical cilia-driven fluid flow. Note that the same embryo was imaged in Figs. 6 and 7 and only the image well water volume differs between the two image acquisition sessions. (a) shows the particle pathline image. (b) shows the two-dimensional, two-component (i.e. v = vxi + vzk) flow velocity field superimposed on the particle pathline image. The arrow direction encodes vector direction, while the arrow color encodes vector magnitude. The recirculatory/vortical whorl noted with a blue arrow in (a) has a faster fluid flow closer to the body than further away. Near the surface of the embryo, the flow is largely anterior-to-posterior (i.e. head-to-tail), while âreturnâ flow current is in a posterior-to-anterior direction. b, body; e, eye; h, head; ps, polystyrene microsphere; t, tail; w, air/water interface. A, anterior; P, posterior.
Fig. 8. Particle residence time in B-scan field of view. The embryo has the same position and orientation as in Fig. 6. The grayscale intensity of the streaks encodes the duration in the field of view. The red rectangle marks the particle that was used for the upper plots in Fig. 9, blue marks the particle used for the lower plots in Fig. 9.
Fig. 9. Plots demonstrating convergence of estimated particle displacement as a function of measurements used in the estimation. The plots on the left side display mean displacement along the x-axis, the right plots mean displacement along the z-axis. The upper two plots are extracted from the pathline in the red rectangle in Fig. 8, the lower two plots are extracted from the pathline in the blue rectangle.
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