Figure 1.
μOCT instrumentation schematic and axial resolution.
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.
Figure 2.
Representative μOCT image of primary HBE culture.
A. A time-averaged (2 s) μOCT image of fully differentiated primary human bronchial epithelial cells derived from a normal subject. From top to bottom, air, mucus layer (mu), PCL layer (pcl; red bar), cilia (green arrows), and epithelial monolayer (ep) are readily seen. ASL depth is defined as the distance between the air-mucus interface and the apical surface of the epithelium (ep). PCL depth is defined by the distance between the ventral surface of the mucus layer and the apical surface of the epithelium. B. H&E stained histology of HBE cells. Scale bar: 10 µm.
Figure 3.
Functional anatomy of excised swine trachea.
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.
Table 1.
μOCT measured parameters from swine trachea ex vivo.
Figure 4.
μOCT functional anatomy of human trachea.
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.
Figure 5.
Comparison of μOCT and gold standard measurements in HBE cells.
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.
Figure 6.
Mucus extrusion from single gland duct.
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.
Figure 7.
μOCT images of ciliary motion pattern in HBE culture and swine trachea.
A. (Top panel) 6-stage schematic of ciliary motion during the recovery stroke; (bottom panel) a μOCT image of fully differentiated primary HBE cells derived from a normal subject shows cilia tips (green) 3–5 µm from the apical cell surface, indicating the recovery stroke. Cilia and mucus are presented in pseudo-colors: green and purple respectively. B. (top panel) 4-stage schematic of ciliary motion during the effective stroke; (bottom panel) μOCT signal of the same cilia after 250 ms that subtend an angle of 114°, delineating an arc with radius of approximately 7 µm during the effective stroke. C. (top panel) 10-stage schematic of ciliary motion during the full ciliary beat cycle; A time-averaged (4 s) image (bottom panel) shows an arc indicating the effective strokes (yellow arrows) and bilobular pattern of the recovery stroke (orange arrows). D. A time-averaged (1 s) image of normal swine trachea shows arcs indicating the effective strokes (yellow arrows) and bilobular pattern suggesting the recovery stroke (orange arrows) in the ciliary motion pattern. Scale bars: 10 µm.
Figure 8.
Cilia motion pattern in cultured HBE cells.
Time-lapsed ciliary motion pattern can be easily identified in the M-mode image (top) of the active epithelial area shown in Figs. 7 A–C and also Movie S5. 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.