Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical Coherence Tomography

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified

μOCT instrumentation schematic and axial resolution.

<p>A. System diagram. RM: reference mirror. OL. objective lens. EC: environmental chamber. AO: analog output board. G: grating. IMAQ: image acquisition board. L: camera lens. LSC: line scan camera. SMF: single mode fiber. PC: personal computer. RAID: redundant array of independent disks. CL: Camera Link cable. B. Depth profile of mirror surface, indicating axial full-width half maximum of 1.3 µm.</p

μOCT measured parameters from swine trachea <i>ex vivo</i>.

<p>Number of samples <i>n</i> refers to separate measurements from a single tissue sample.</p

Functional anatomy of excised swine trachea.

<p>A. μOCT image. Yellow bar indicates airway surface liquid (ASL) depth and Red bar indicates PCL depth. Epithelium (ep) and lamina propria (lp) are also visible. B. H&E stained histology image illustrating cilia (c), epithelium (ep) and lamina propria (lp). Scale bar of both images: 10 µm.</p

Mucus extrusion from single gland duct.

<p>A. A representative frame from a μOCT video of trachea dissected from a swine shows mucus (yellow arrow) extrusion from a gland duct (gd) in lamina propria (lp). B. Three-dimensional reconstructed en face view allows estimation of luminal area of the duct. In swine trachea, mucus extrusion rate is 0.095 nL/min (N = 3, ±SEM = ±0.006) at room temperature.</p

Comparison of μOCT and gold standard measurements in HBE cells.

<p>All error bars represent SEM. A. ASL depth measured with μOCT (7.40±1.82 µm, n = 5) and confocal microscopy (7.76±0.87 µm, n = 6). B. CBF measured with μOCT (9.32±0.27 Hz, n = 4) and Hoffman contrast microscopy (10.17±0.56 Hz, n = 4). C. MCT velocity measured with μOCT (24.22±14.88 µm/sec, n = 6) and particle-tracking fluorescence microscopy (1.91±0.62 µm/sec, n = 11). Number of measurements n refers to separate wells analyzed.</p

Cilia motion pattern in cultured HBE cells.

<p>Time-lapsed ciliary motion pattern can be easily identified in the M-mode image (top) of the active epithelial area shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054473#pone-0054473-g007" target="_blank">Figs. 7 A–C</a> and also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054473#pone.0054473.s005" target="_blank">Movie S5</a>. The continuity of the sinusoidal pattern in the M-mode image indicates that the entire beat cycle was captured. Corresponding time-lapse intensity analysis (bottom) reveals triphasic pattern of the ciliary beat cycle: the recovery stroke (blue line), the effective stroke (orange line) and the rest phase in between the effective stroke and next effective stroke.</p

μOCT functional anatomy of human trachea.

<p>A. A time-averaged (1 s) μOCT image of human tracheal tissue shows epithelium (ep), lamina propria (lp), gland duct (gd), mucus (m), cilia (ci) and goblet cells (gc). Yellow bar indicates airway surface liquid (ASL) depth and Red bar indicates PCL depth. B. Orthogonal view at the position indicated by the dashed blue line shows the whole gland duct and the goblet cell in A. Ciliary beat pattern and possible goblet cell nucleus are clearly seen in the inset. Scale bars: 20 µm.</p