Ex vivo visualization of human ciliated epithelium and quantitative analysis of induced flow dynamics by using optical coherence tomography

Abstract

Background and objective: Cilia-driven mucociliary clearance is an important self-defense mechanism of great clinical importance in pulmonary research. Conventional light microscopy possesses the capability to visualize individual cilia and its beating pattern but lacks the throughput to assess the global ciliary activities and flow dynamics. Optical coherence tomography (OCT), which provides depth-resolved cross-sectional images, was recently introduced to this area.

Materials and methods: Fourteen de-identified human tracheobronchial tissues are directly imaged by two OCT systems: one system centered at 1,300 nm with 6.5 μm axial resolution and 15 μm lateral resolution, and the other centered at 800 nm with 2.72 μm axial resolution and 5.52 μm lateral resolution. Speckle variance images are obtained in both cross-sectional and volumetric modes. After imaging, sample blocks are sliced along the registered OCT imaging plane and processed with hematoxylin and eosin (H&E) stain for comparison. Quantitative flow analysis is performed by tracking the path-lines of microspheres in a fixed cross-section. Both the flow rate and flow direction are characterized.

Results: The speckle variance images successfully segment the ciliated epithelial tissue from its cilia-denuded counterpart, and the results are validated by corresponding H&E stained sections. A further temporal frequency analysis is performed to extract the ciliary beat frequency (CBF) at cilia cites. By adding polyester microspheres as contrast agents, we demonstrate ex vivo imaging of the flow induced by cilia activities of human tracheobronchial samples.

Conclusion: This manuscript presents an ex vivo study on human tracheobronchial ciliated epithelium and its induced mucous flow by using OCT. Within OCT images, intact ciliated epithelium is effectively distinguished from cilia-denuded counterpart, which serves as a negative control, by examining the speckle variance images. The cilia beat frequency is extracted by temporal frequency analysis. The flow rate, flow direction, and particle throughput are obtained through particle tracking. The availability of these quantitative parameters provides us with a powerful tool that will be useful for studying the physiology, pathophysiology and the effectiveness of therapies on epithelial cilia function, as well as serve as a diagnostic tool for diseases associated with ciliary dysmotility. Lasers Surg. Med. 49:270-279, 2017. © 2017 Wiley Periodicals, Inc.

Keywords: flow dynamics; human tracheobronchial tissue; motile cilia; mucociliary clearance; optical coherence tomography; speckle variance.

Figure 1

Human tracheobronchial tissue OCT images (a), (c), (e), and (g) and corresponding H&E stained tissue section (b), (d), (f), and (h). The samples represented in (a), (c), (e), (g) were sample 8, sample 1, sample 8, and sample 3, respectively. The OCT images were imaged by Thorlabs Telesto system. BM: Basement membrane, C: Cartilage ring. CE: Ciliated Epithelium, M: Mucus. MG: Mucus secreting glands. MD: Mucus gland duct. PC: Perichondrium.

Figure 2

Observation of mucus excretion and its effect. Mucus secreted from the mucus gland duct (sample 2). The images were imaged by Thorlabs Telesto system. M: Mucus. MD: Mucus gland duct. T: Tracheal wall.

Figure 3

The calculated speckle variance image in comparison with its original intensity OCT image as well as corresponding H&E stained tissue (sample 8). (a) H&E stained tissue histology slide. (b) The intensity OCT image. (c) Calculated speckle variance image, where the ciliated epithelium has a higher variance than other parts of the sample. It is visualized as a layer on top of the specimen. Two zoom-in regions (d) and (f) of the speckle variance image and their corresponding zoomed-in H&E stained tissue (e) and (g). In (d), the entire specimen is covered by ciliated epithelium as confirmed in (e). In (f), there are two regions, where no high contrast layer-like structure is presented. Those two regions are not covered by cilia, as verified by the H&E stained tissue (g). The images were imaged by Thorlabs Telesto system. NC: cilia-denuded. C: ciliated epithelium. Note: the protrusions that C1 pointed to in (f) is an interpolation artifact instead of cilia. The cilia are represented by the high intensity layer beneath.

Figure 4

Three-dimensional visualization of human tracheal specimen (sample 9). The volumetric dataset consists of 500 pixels (width) × 45 pixels (length) × 1024 pixels (depth) and was obtained by using the customized scanning protocol with high resolution SDOCT detailed in the method section. (a) Structural image overlaid against threshold variance image. The ciliary motion is only observed on the right half surface of the sample (b) The cross-sectional view of the sample, and (c) its corresponding variance image. M: mucus. CE: ciliated epithelium, which is visible in speckle variance image. T: trachea.

Figure 5

The temporal frequency analysis on the ciliary beat frequency of human trachea sample with the presence of mucus layer (sample 13). (a) A color-coded spatial map of dominant frequency amplitude after thresholding and normalization. The pseudo-color spatial map was overlaid with the original intensity OCT image. (b) Speckle variance image. (c) A color-coded spatial map of dominant frequency. The pseudo-color spatial map was overlaid with the original intensity OCT image. (d) The temporal spectra of two locations. The red curve represents the ciliated epithelium, and the blue curve represents the cilia-denuded epithelium. We observed an 8 Hz peak on ciliated epithelium. The original intensity OCT image was averaged 4 times. All images were taken by the custom-built high-resolution SD-OCT system. CE: ciliated epithelium. CD: ciliadenuded Epithelium.

Figure 6

The flow images of the microspheres induced by the beating of ciliated epithelium. (a) and (c) are MIP image by integrating all 200 B-scans. (b) and (d) are corresponding color-encoded time-elapsed images. The sample represented in (a), (c) are sample 5 and sample 6, respectively. The time span of all the images are 16.627 s, while the 1st eighth interval of the entire time duration is colored by red, the 2nd of that is colored by yellow, …, and 8th of that is colored by purple. The images were imaged by Thorlabs Telesto system.

Figure 7

The quantitative cilia-driven flow analysis (sample 5). The dataset is the same as that in Figure 5 (a). (a) The vector visualization of flow dynamics. The direction of the arrow represents the averaged flow direction and the flow rate is encoded in both arrow length and color. (b) The histogram of all the tracked particles inside the red box shown in (a). The total time duration is 16.627 s and 2,251 microspheres are tracked. The images were imaged by Thorlabs Telesto system.

Figure 8

The MIP image of microsphere flow at an orthogonal direction to superior-inferior axis of trachea tissue (sample 5). No particle path-line is presented. The left half of the sample has much higher microsphere intensity than right half, which implies the ciliary motion is stronger in the left half than that of the right half. The images were imaged by Thorlabs Telesto system.