Chronic dysregulated influx of neutrophil into the airway increases neutrophil burden and augments the inflammatory processes often observed in diseases such as cystic fibrosis. The quantification of neutrophil influx is often accomplished with the use of destructive tests such as imaging cytometry and myeloperoxidase assay. However, those methods are unable to capture information about the cascade of events that precede trans-epithelium migration. In this work, we employed a high resolution micro-optical coherence tomography (µOCT) technology to perform real time imaging of neutrophil activity across airway epithelial cells grown on the underside of Transwell permeable supports. This inverted configuration allows the creation of an air-liquid interface at the apical side of the cells. The µOCT imaging technology, based on the principles of spectral-domain OCT, has a lateral and axial resolution of 2 and 1.3µm, respectively. In addition, it has an axial range of approximately 300µm and is capable of recording cross-sectional images at 40 fps. By raster scanning the illumination beam, the behavior of the neutrophils across a 3D volume can be recorded over time. Thus, this imaging modality is capable of resolving individual neutrophils and, potentially, capturing the cascading events involving neutrophil tethering, subsequent adhesion to activated epithelial cells and the ultimate passage through the epithelial cells to the air space on the apical side. As a result, not only can the amount of neutrophil migration be quantified, how neutrophils behave, organize and interact with the epithelial cells and each other can also be more closely analyzed by µOCT imaging.

Mucociliary flow is an important defense mechanism in the lung to remove inhaled pathogens and pollutants. A disruption of ciliary flow can lead to respiratory infections. Even though patients in the intensive care unit (ICU) either have or are very susceptible to respiratory infections, mucociliary flow is not well understood in the ICU setting. We recently demonstrated that hyperoxia, a consequence of administering supplemental oxygen to a patient in respiratory failure, can lead to a significant reduction of cilia-driven fluid flow in mouse trachea. There are other factors that are relevant to ICU medicine that can damage the ciliated tracheal epithelium, including inhalation injury and endotracheal tube placement. In this study we use two animal models, Xenopus embryo and ex vivo mouse trachea, to analyze flow defects in the injured ciliated epithelium. Injury is generated either mechanically with a scalpel or chemically by calcium chloride (CaCl2) shock, which efficiently but reversibly deciliates the embryo skin. In this study we used optical coherence tomography (OCT) and particle tracking velocimetry (PTV) to quantify cilia driven fluid flow over the surface of the Xenopus embryo. We additionally visualized damage to the ciliated epithelium by capturing 3D speckle variance images that highlight beating cilia. Mechanical injury disrupted cilia-driven fluid flow over the injured site, which led to a reduction in cilia-driven fluid flow over the whole surface of the embryo (n=7). The calcium chloride shock protocol proved to be highly effective in deciliating embryos (n=6). 3D speckle variance images visualized a loss of cilia and cilia-driven flow was halted immediately after application. We also applied CaCl2-shock to cultured ex vivo mouse trachea (n=8) and found, similarly to effects in Xenopus embryo, an extensive loss of cilia with resulting cessation of flow. We investigated the regeneration of the ciliated epithelium after an 8 day incubation period, and found that cilia had regrown and flow was completely restored. In conclusion, OCT is a valuable tool to visualize injury of the ciliated epithelium and to quantify reduction of generated flow. This method allows for systematic investigation of focal and diffuse injury of the ciliated epithelium and the assessment of mechanisms to compensate for loss of flow.

Failure in mucociliary clearance is responsible for severe diseases like cystic fibroses, primary ciliary dyskinesia or asthma. Visualizing the mucous transport in-vivo will help to understanding transport mechanisms as well as developing and validating new therapeutic intervention. However, in-vivo imaging is complicated by the need of high spatial and temporal resolution. Recently, we developed microscopy optical coherence tomography (mOCT) for non-invasive imaging of the liquid-air interface in intact murine trachea from its outside. Whereas axial resolution of 1.5 µm is achieved by the spectral width of supercontinuum light source, lateral resolution is limited by aberrations caused by the cylindric shape of the trachea and optical inhomogenities of the tissue. Therefore, we extended our mOCT by a deformable mirror for compensation of the probe induced aberrations. Instead of using a wavefront sensor for measuring aberrations, we harnessed optimization of the image quality to determine the correction parameter. With the aberration corrected mOCT ciliary function and mucus transport was measured in wild type and βENaC overexpressing mice, which served as a model for cystic fibrosis.

The ciliated epithelium is important to the human respiratory system because it clears mucus that contains harmful microorganisms and particulate matter. We report the ex vivo visualization of human trachea/bronchi ciliated epithelium and induced flow characterized by using spectral-domain optical coherence tomography (SD-OCT). A total number of 17 samples from 7 patients were imaged. Samples were obtained from Columbia University Department of Anesthesiology’s tissue bank. After excision, the samples were placed in Gibco Medium 199 solution with oxygen at 4°C until imaging. The samples were maintained at 36.7°C throughout the experiment. The imaging protocol included obtaining 3D volumes and 200 consecutive B-scans parallel to the head-to-feet direction (superior-inferior axis) of the airway, using Thorlabs Telesto system at 1300 nm at 28 kHz A-line rate and a custom built high resolution SDOCT system at 800nm at 32 kHz A-line rate. After imaging, samples were processed with H and E histology. Speckle variance of the time resolved datasets demonstrate significant contrast at the ciliated epithelium sites. Flow images were also obtained after injecting 10μm polyester beads into the solution, which shows beads traveling trajectories near the ciliated epithelium areas. In contrary, flow images taken in the orthogonal plane show no beads traveling trajectories. This observation is in line with our expectation that cilia drive flow predominantly along the superior-inferior axis. We also observed the protective function of the mucus, shielding the epithelium from the invasion of foreign objects such as microspheres. Further studies will be focused on the cilia’s physiological response to environmental changes such as drug administration and physical injury.

The human respiratory system is protected by a defense mechanism termed mucociliary clearance (MCC). Deficiency in MCC leads to respiratory obstruction and pulmonary infection, which often are the main causes of morbidity and mortality in diseases such as cystic fibrosis and chronic obstructive pulmonary disease (COPD). Studying key parameters that govern MCC, including ciliary beat frequency, velocity and volume of airway mucus transport, as well as periciliary liquid layer thickness are therefore of great importance in understanding human respiratory health. However, direct, in vivo visualization of ciliary function and MCC has been challenging, hindering the diagnosis of disease pathogenesis and mechanistic evaluation of novel therapeutics. Our laboratory has previously developed a 1-µm resolution optical coherence tomography method, termed Micro-OCT, which is a unique tool for visualizing the spatiotemporal features of ciliary function and MCC. We have previously described the design of a flexible 2.5 mm Micro-OCT probe that is compatible with standard flexible bronchoscopes. This device utilizes a common-path interferometer and annular sample arm apodization to attain a sharply focused spot over an extended depth of focus. Here, we present the most recent iteration of this probe and demonstrate its imaging performance in a mouse trachea tissue culture model. In addition, we have developed an ergonomic assembly for attaching the probe to a standard bronchoscope. The ergonomic assembly fixes the Micro-OCT probe’s within the bronchoscope and contains a means transducing linear motion through the sheath so that the Micro-OCT beam can be scanned along the trachea. We have tested the performance of these devices for Micro-OCT imaging in an anatomically correct model of the human airway. Future studies are planned to use this technology to conduct Micro-OCT in human trachea and bronchi in vivo.

In healthy humans, the physiological state in the distal lung alveolar acinar units is tightly regulated by normal homeostatic mechanisms. Pulmonary abnormalities such as chronic obstructive pulmonary disease, that are characterized by recurrent cycles of inflammation and infection involving dense infiltration by myeloid derived peripheral blood cells, may result in significant perturbation of the homeostatic baselines of physiology in addition to host tissue damage. Therefore, the ability to quantify and monitor physiology (e.g. pH, glucose level, oxygen tension) within the alveolar acinar units would provide a key biomarker of distal lung innate defence. Although in vitro modeling of fundamental biological processes show remarkable sensitivity to physiological aberrations, little is known about the physiological state of the distal lung due to the inability to concurrently access the alveolar sacs and perform real-time sensing. Here we report on previously unobtainable measurements of alveolar pH using a fiber-optic optrode and surface enhanced Raman spectroscopy (SERS) and show that alveolar pH changes in response to ventilation. The endoscope-deployable optrode consisted of para-mercaptobenzoic acid functionalized 150 nm gold nanoshells located at the distal end, and an asymmetric dual-core optical fiber designed for spatially separated optical pump delivery and SERS signal collection in order to circumvent the unwanted Raman signal originating from the fiber itself. We demonstrate a ~ 100-fold increase in SERS signal-to-fiber background ratio and pH sensing at multiple sites in the respiratory acinar units of a whole ex vivo ovine lung model with a measurement accuracy of ± 0.07 pH units.

Granulation and tumor regrowth in the area of bronchi stent implants may result in restenosis. It had been shown that by means of Thulium-Fibre-Laser (TFL) a controlled ablation and reduction of the tissue within the stent could be performed. When using Nd:YAG irradiation there is risk for explosive flames, burns of fibre and stent, ruptures of stent meshes as well as perforation of stent and cover. Therefore it was the aim to investigate the safety margin when using TFL. Four different types of clinical used stents (with/without cover) were fixed to pig trachea tissue. Irradiation was performed by fibre assisted TFL-1940nm-laser irradiation while laser power, light application duration and distance, as well as oxygen percentage and contamination were varied. In case of Nitinol-stents rupture were observed at power levels ≥7W or distances of <5mm, oxygen conc. of 40% result in increased flame appearance. Polyurethan-covers were ruptured at each variable, flame appeared at 5W. Silicon-stents were destroyed at power levels of about 5W and distances of <5mm and additionally 30%-oxygen or contamination either by blood or soot result in increased appearance of burns and flames. Based upon these observations in clinical TFL-irradiation the distance should ≥5 mm and the power level should be ≤6W. Furthermore the oxygen conc. should not exceed 30% and short term continuous irradiation of less than 15s exposition should be considered. In case of Silicon-stents light application on contaminated area should be avoided.

Antibiotic resistance is a serious global concern. One way to tackle this problem is to develop new and sensitive approaches to diagnose bacterial infections and prevent unnecessary antibiotic use. With recent developments in optical molecular imaging, we are one step closer to in situ rapid detection of bacterial infections. We present here bespoke fluorescent probes for bacterial detection in ex vivo human lung tissue using fluorescence lifetime imaging microscopy (FLIM). Two in-house synthesised bespoke probes were used in this study to detect and differentiate between Gram positive and Gram negative bacterial strain using their fluorescence lifetime in the ex vivo human lung tissue. The average fluorescence lifetime of Gram positive probe (n=12) was 2.40 ± 0.25 ns and Gram negative (n=12) was 6.73 ± 0.49 ns. The human lung tissue (n=12) average fluorescence lifetime value was found to be 3.43 ± 0.19 ns. Furthermore we were also able to distinguish between dead or alive bacteria in ex vivo lung tissue based on difference in their lifetime. We have developped Fibre-FLIM methods to enable clinical translation within the Proteus Project (www.proteus.ac.uk).

The health of the tracheal mucosa is an important, but poorly understood, aspect of critical care medicine. Many critical care patients are mechanically ventilated through an endotracheal tube that can cause local inflammation and blunt damage to the ciliated epithelial cells lining the trachea. These cilia clear mucus and infectious agents from the respiratory tract, so impaired ciliary function may lead to increased susceptibility to respiratory infection. Therefore, a minimally-invasive method to monitor mucosal health and ciliary function in intubated patients would be valuable to critical care medicine. Optical metabolic imaging (OMI) can quantitatively assess the metabolic state of cells by measuring the fluorescence intensities of endogenous metabolic co-enzymes NAD(P)H and FAD. OMI is especially attractive for assessing tracheal health because OMI is label-free, and ciliary function is tightly linked to the levels of NAD(P)H and FAD. In this study, we apply widefield OMI to ex vivo mouse tracheae (n=6), and demonstrate that the optical redox ratio (fluorescence intensity of NAD(P)H divided by the intensity of FAD) is sensitive to changes in the cellular metabolism of the tracheal mucosa. We observed a 46% increase in the redox ratio 20 minutes after treatment with 10mM of sodium cyanide (p<0.001, 95% CI [40%, 52%]), an inhibitor of oxidative cellular respiration. In addition to being a proof-of-concept demonstration, Pseudomonas aeruginosa, an important cause of morbidity and mortality in CF patients and in the ICU, produces hydrogen cyanide. Our results support the development of minimally-invasive fiber-optic probes for in vivo monitoring of tracheal health.