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 1300 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.

Visualizing flow in an intact CSF network using optical coherence tomography: implications for human congenital hydrocephalus

“…Importantly, studies in other organ systems with cilia-lined fluid cavities (e.g., the fallopian tube or upper respiratory tract) have revealed the importance of the interaction between the cilia, chamber geometry, and fluid viscosity on the mechanical polarization of the fluid-flow. This is a severe limitation for explant experiments that are widely used to understand the polarized CSF circulation 16–19 . Therefore, studying explants of only one chamber of a post-mortem ventricle is suboptimal for analyzing the CSF flow network.…”

Frequency-constrained robust principal component analysis: a sparse representation approach to segmentation of dynamic features in optical coherence tomography imaging

“…Though not explicitly compared, the results additionally demonstrate clear advantages of the technique over the presently used Speckle Variance method. The major drawbacks of SV processing are that the technique is blind to the source of any temporally varying signal, is influenced by noise artifacts and low SNR regions, and significant parameter tuning is required to produce adequate identification of the moving object of interest [17][18][19][20][21][22]. Inspection of the quantitative results shown in Table 1 and the Fig.…”

“…Individual cilia are too small to directly observe their motion with most OCT systems [18]; however, their motion produces speckle-like fluctuations in the OCT image intensity that can be detected using SV imaging [19]. As a result, this property has been utilized in many studies to extract quantitative information on cilia behavior [20][21][22]. SV imaging, however, lacks sensitivity to other types of motion, and relies heavily on subjective processing which is prohibitive in studies involving large volumes of data.…”

“…Data was acquired using a custom Ultra-High Resolution OCT (UHR-OCT) system designed in house that has previously been used for imaging human trachea samples [22,24,25]. The UHR system operates at a frame rate of 64 Hz with 2.72 µm axial resolution and 5.52 µm lateral resolution.…”

Label-free intratissue activity imaging of alveolar organoids with dynamic optical coherence tomography

“…Several DOCT methods have been demonstrated to visualize the intratissue and intracellular activities through the fluctuation magnitude [ 28 – 30 ], time-frequency spectrum [ 28 , 31 – 34 ], and time-correlation property [ 30 , 35 , 36 ] of OCT signals. DOCT has been applied to a variety of samples including human esophageal and cervical biopsies [ 34 ], ex vivo mouse or rat organs [ 29 , 31 , 33 , 36 – 38 ], in vivo zebrafish [ 39 ], in vitro cell culture [ 40 ], spheroid [ 30 , 41 ], mammaria organoid [ 42 ], and retinal organoid [ 43 ].…”