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Dynamic microscopic optical coherence tomography to visualize the morphological and functional micro-anatomy of the airways

Tabea Kohlfaerber, Mario Pieper, Michael Münter, Cornelia Holzhausen, Martin Ahrens, Christian Idel, Karl-Ludwig Bruchhage, Anke Leichtle, Peter König, Gereon Hüttmann, and Hinnerk Schulz-Hildebrandt

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Abstract

In the imaging of airway tissue, optical coherence tomography (OCT) provides cross-sectional images of tissue structures, shows cilia movement and mucus secretion, but does not provide sufficient contrast to differentiate individual cells. By using fast sequences of microscopic resolution OCT (mOCT) images, OCT can use small signal fluctuations to overcome lack in contrast and speckle noise. In this way, OCT visualizes airway morphology on a cellular level and allows the tracking of the dynamic behavior of immune cells, as well as mucus transport and secretion. Here, we demonstrate that mOCT, by using temporal tissue fluctuation as contrast (dynamic mOCT), provides the possibility to study physiological and pathological tissue processes in vivo.

© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement

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Supplementary Material (5)

NameDescription
Visualization 1       Visualization 1 shows the increase in ciliary beat frequency after administration of ATP-gamma-S. It shows the entire time-lapse image sequence of the dmOCT-B scan shown in Fig. 3(a). ATP-gamma-S was applied with a pipette at t = 05:20 minutes.
Visualization 2       Visualization 2 shows in the same image series than in Visualization 1 the corresponding changes in the dynamic contrast after administration of ATP-gamma-S at t = 05:20 minutes.
Visualization 3       Visualization 3 correlates to Fig. 3(c) and shows the entire time-lapse image sequence of dmOCT B-scans. Mucus secretion starts after ATP-gamma-S application at second 45.
Visualization 4       Visualization 4 correlates to Fig.4(a,b). Dynamic mOCT (on top) and averaged mOCT (at the bottom) B-scans are shown for the entire time-lapse image sequence. The whole sample is shown to the left and two magnifications are to the right. The position
Visualization 5       Visualization 5 correlates to Fig.4(c,d). Dynamic mOCT (left) and averaged mOCT (right) B-scans are shown for the entire time-lapse image sequence. Magnification correlates to Fig.4 (d) and is marked by a red square.

Data Availability

Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.

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Figures (4)
Fig. 1.
Fig. 1. (a) Schematic mOCT setup. FC: 50/50 fiber coupler; C1/C2: collimators; Gx, Gy: galvanometer mirror scanners; L1, L2: beam expander lenses; L3: microscope objective; DC: dispersion compensation; RR: retroreflector; DAQ, data acquisition device; PC: computer for data acquisition and scanning control.

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Fig. 2.
Fig. 2. Images of ex vivo mouse trachea from one acquired volume. (a) Exemplary semi thin section stained with methylene blue-azure II from a murine trachea. (b) Dynamic mOCT B-scan and (c) corresponding averaged mOCT B-scan. (d-k) En face images of the same tissue at different planes marked red in the preceding B-scans. Dynamic contrast is shown in the center line (d-g) and the corresponding averaged mOCT image below (h-k). (d,h) Cilia, (e,i) epithelium, (f,j) connective tissue layer with immune cells (marked by red arrows) (g,k) connective tissue, cartilage (red star) and lymphatic vessel (yellow star) with valve (marked by yellow arrow).

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Fig. 3.
Fig. 3. Gland secretory ducts in two ex vivo mouse tracheas. B-scans prior to and after ATP-$\gamma$-S stimulation are shown. (a) Dynamic mOCT B-scans, (b) corresponding dominant frequencies of segmented cilia superimposed on the averaged gray scale image. Different cilia are indicated by a red, white and black arrow for further detailed frequency analysis. (c) Dynamic mOCT B-scan of a different sample during secretion (d) corresponding averaged mOCT B-scan. Entire time-lapse image sequence is shown in Visualization 1, Visualization 2 and Visualization 3. Time (t) is shown in minutes:seconds format.

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Fig. 4.
Fig. 4. Cell migration in airway tissue. (a) Cross-sectional dmOCT and (b) averaged mOCT images of ex vivo mouse trachea (see Visualization 4). (c) Series of images from the ROI indicated by the yellow square in (a) and (b) show the progression of an immune cell, indicated by red arrows at 5 different time points (mOCT on the top and dmOCT at the bottom). (d) Cross-sectional dmOCT, (e) averaged standard mOCT image and (f) H&E stained histological section of ex vivo human nasal concha. Red arrow indicates immune cells in the epithelial layer (Visualization 5). (g) series of images from ROI indicated in (d) and (e) by the red square shows an appearing and disappearing immune cell in the imaged B-scan layer (red arrow). The migration of two individual immune cells is indicated by a white and black star at 5 different time points (t). Scale bar (c,g) represents 20 $\mathrm{\mu}$m. Time (t) is shown in minutes:seconds.

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