Ute A. Gamm

Quantitative optical coherence tomography imaging of intermediate flow defect phenotypes in ciliary physiology and pathophysiology

Cilia-driven fluid flow is a critical yet poorly understood aspect of pulmonary physiology. Here, we demonstrate that optical coherence tomography-based particle tracking velocimetry can be used to quantify subtle variability in cilia-driven flow performance in Xenopus, an important animal model of ciliary biology. Changes in flow performance were quantified in the setting of normal development, as well as in response to three types of perturbations: mechanical (increased fluid viscosity), pharmacological (disrupted serotonin signaling), and genetic (diminished ciliary motor protein expression). Of note, we demonstrate decreased flow secondary to gene knockdown of kif3a, a protein involved in ciliogenesis, as well as a dose-response decrease in flow secondary to knockdown of dnah9, an important ciliary motor protein.

Three-dimensional, three-vector-component velocimetry of cilia-driven fluid flow using correlation-based approaches in optical coherence tomography

Microscale quantification of cilia-driven fluid flow is an emerging area in medical physiology, including pulmonary and central nervous system physiology. Cilia-driven fluid flow is most completely described by a three-dimensional, three-component (3D3C) vector field. Here, we generate 3D3C velocimetry measurements by synthesizing higher dimensional data from lower dimensional measurements obtained using two separate optical coherence tomography (OCT)-based approaches: digital particle image velocimetry (DPIV) and dynamic light scattering (DLS)-OCT. Building on previous work, we first demonstrate directional DLS-OCT for 1D2C velocimetry measurements in the sub-1 mm/s regime (sub-2.5 inch/minute regime) of cilia-driven fluid flow in Xenopus epithelium, an important animal model of the ciliated respiratory tract. We then extend our analysis toward 3D3C measurements in Xenopus using both DLS-OCT and DPIV. We demonstrate the use of DPIV-based approaches towards flow imaging of Xenopus cerebrospinal fluid and mouse trachea, two other important ciliary systems. Both of these flows typically fall in the sub-100 μm/s regime (sub-0.25 inch/minute regime). Lastly, we develop a framework for optimizing the signal-to-noise ratio of 3D3C flow velocity measurements synthesized from 2D2C measures in non-orthogonal planes. In all, 3D3C OCT-based velocimetry has the potential to comprehensively characterize the flow performance of biological ciliated surfaces.

Particle streak velocimetry-optical coherence tomography: a novel method for multidimensional imaging of microscale fluid flows.

We present a new OCT method for flow speed quantification and directional velocimetry: particle streak velocimetry-OCT (PSV-OCT). PSV-OCT generates two-dimensional, 2.5-vector component (vx ,|vy |,vz ) maps of microscale flow velocity fields. Knowledge of 2.5-vector components also enables the estimation of total flow speed. The enabling insight behind PSV-OCT is that tracer particles in sparsely-seeded fluid flow trace out streaks in (x,z,t)-space. The streak orientations in x-t and z-t yield vx and vz , respectively. The in-plane (x-z plane) residence time yields the out-of-plane speed |vy |. Vector component values are generated by fitting streaks to a model of image formation that incorporates equations of motion in 3D space. We demonstrate cross-sectional estimation of (vx ,|vy |,vz ) in two important animal models in ciliary biology: Xenopus embryos (tadpoles) and mouse trachea.

Quantifying hyperoxia-mediated damage to mammalian respiratory cilia-driven fluid flow using particle tracking velocimetry optical coherence tomography

Oxygen supplementation [hyperoxia, increased fraction of inspired oxygen (FiO 2 )] is an indispensable treatment in the intensive care unit for patients in respiratory failure. Like other treatments or drugs, hyperoxia has a risk-benefit profile that guides its clinical use. While hyperoxia is known to damage respiratory epithelium, it is unknown if damage can result in impaired capacity to generate cilia-driven fluid flow. Here, we demonstrate that quantifying cilia-driven fluid flow velocities in the sub-100 μm/s regime (sub-0.25 in./min regime) reveals hyperoxia-mediated damage to the capacity of ciliated respiratory mucosa to generate directional flow. Flow quantification was performed using particle tracking velocimetry optical coherence tomography (PTV-OCT) in ex vivo mouse trachea. The ability of PTV-OCT to detect biomedically relevant flow perturbations in the sub-100 μm/s regime was validated by quantifying temperature- and drug-mediated modulation of flow performance in ex vivo mouse trachea. Overall, PTV-OCT imaging of cilia-driven fluid flow in ex vivo mouse trachea is a powerful and straightforward approach for studying factors that modulate and damage mammalian respiratory ciliary physiology.

Ex vivo visualization of human ciliated epithelium and quantitative analysis of induced flow dynamics by using optical coherence tomography: EX VIVO VISUALIZATION OF HUMAN CILIATED EPITHELIUM

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.

Optical coherence tomography and autofluorescence microscopy of respiratory ciliated epithelia (Conference Presentation)

Efficient mucociliary clearance is necessary to protect the respiratory tract from infection. Mucociliary dysfunction is common in respiratory diseases including asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Rescuing mucociliary clearance by stimulating the metabolism of respiratory ciliated epithelia could offer new treatments for respiratory diseases. However, the coupling between cellular metabolism and mechanical output in respiratory ciliated epithelia is poorly understood. We propose to study this coupling with autofluorescence microscopy and optical coherence tomography (OCT), to measure cellular metabolism and ciliary motility, respectively. The autofluorescent metabolic co-enzymes NAD(P)H and FAD provide non-invasive measures of metabolism through the optical redox ratio (NAD(P)H intensity divided by FAD intensity), while OCT measures both the frequency of ciliary beating and cilia-driven fluid flow. Preliminary experiments were performed in ex vivo mouse trachea using an epifluorescence microscope and a spectral-domain OCT system. Cilia-driven fluid flow was quantified using 2D particle tracking velocimetry (PTV-OCT) and TrackMate, a particle-tracking tool. PTV-OCT was validated by manual particle tracking (within 4% agreement) and a calibrated flow phantom (r=0.998, p<0.001). Treatment of the trachea with cyanide, a complex IV inhibitor that reduces intracellular ATP levels, demonstrated that an increase in optical redox ratio (p<0.001) reflects a decrease in cilia-driven flow (p<0.05). Additional studies using human samples are underway to explore how pathologically altered metabolism affects ciliary motility. This optical imaging approach could provide a better understanding of respiratory disease pathogenesis, and new therapeutic targets. In the future, these technologies could also monitor intensive care patients through an endoscope.

Visualization and quantification of injury to the ciliated epithelium using quantitative flow imaging and speckle variance optical coherence tomography

Mucociliary flow is an important defense mechanism in the lung to remove inhaled pathogens and pollutants. Disruption of ciliary flow can lead to respiratory infections. Multiple factors, from drugs to disease can cause an alteration in ciliary flow. However, less attention has been given to injury of the ciliated epithelium. In this study, we show how optical coherence tomography (OCT) can be used to investigate injury to the ciliated epithelium in a multi-contrast setting. We used particle tracking velocimetry (PTV-OCT) to investigate the cilia-driven flow field and 3D speckle variance imaging to investigate size and extent of injury caused to the skin of Xenopus embryos. Two types of injuries are investigated, focal injury caused by mechanical damage and diffuse injury by a calcium chloride shock. We additionally investigate injury and regeneration of cilia to calcium chloride on ex vivo mouse trachea. This work describes how OCT can be used as a tool to investigate injury and regeneration in ciliated epithelium.

Functional optical imaging of tracheal health (Conference Presentation)

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.

Quantification and visualization of injury and regeneration in the developing ciliated epithelium using quantitative flow imaging and speckle variance optical coherence tomography (Conference Presentation)

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.

Particle streak velocimetry-OCT (PSV-OCT): a novel method for multi-vector component velocimetry of microscale flow physiology(Conference Presentation)

We present a new method for 2.5 and 3 vector component velocimetry. We call this method particle streak velocimetry OCT (PSV-OCT). PSV-OCT generates two-dimensional, 2.5 vector component (v_x,|v_y|,v_z) cross-sectional maps of microscale flow velocity (e.g. biological cilia-driven fluid flow). The enabling insight is that a tracer particle in sparsely-seeded fluid flow traces out streaks in (x,z,t)-space. The streak orientations in x-t and z-t yield v_x and v_z, respectively. The in-plane (x-z plane) residence time yields the out-of-plane speed |v_y|. Vector component values are generated by fitting streaks to a model of image formation. We demonstrate cross-sectional estimation of (v_x,|v_y|,v_z) in two important animal models in ciliary biology: Xenopus embryos (tadpoles) and mouse trachea. Further, by incorporation the assumption of incompressible flow into the estimation process, we are able to generate 3 vector component (v_x,v_y,v_z) estimates in three spatial dimensions from 2.5 vector component measurements taken in parallel OCT planes in 3D space.