Metachrony drives effective mucociliary transport via a calcium-dependent mechanism.

The mucociliary transport apparatus is critical for maintaining lung health via the coordinated movement of cilia to clear mucus and particulates. A metachronal wave propagates across the epithelium when cilia on adjacent multiciliated cells beat slightly out of phase along the proximal-distal axis of the airways in alignment with anatomically directed mucociliary clearance. We hypothesized that metachrony optimizes mucociliary transport (MCT) and that disruptions of calcium signaling would abolish metachrony and decrease MCT. We imaged bronchi from human explants and ferret tracheae using micro-Optical Coherence Tomography (µOCT) to evaluate airway surface liquid depth (ASL), periciliary liquid depth (PCL), cilia beat frequency (CBF), MCT, and metachrony in situ. We developed statistical models that included covariates of MCT. Ferret tracheae were treated with BAPTA-AM (chelator of intracellular Ca2+), lanthanum chloride (nonpermeable Ca2+channel competitive antagonist), and repaglinide (inhibitor of calaxin) to test calcium-dependence of metachrony. We demonstrated metachrony contributes to mucociliary transport of human and ferret airways. MCT was augmented in regions of metachrony compared to non-metachronous regions by 48.1%, P=0.0009 or 47.5%, P<0.0020 in humans and ferrets, respectively. PCL and metachrony were independent contributors to MCT rate in humans; ASL, CBF, and metachrony contribute to ferret MCT rates. Metachrony can be disrupted by interference with calcium signaling including intracellular, mechanosensitive channels, and calaxin. Our results support that the presence of metachrony augments MCT in a calcium-dependent mechanism.

Next-generation endoscopic probe for detection of esophageal dysplasia using combined OCT and angle-resolved low-coherence interferometry.

Angle-resolved low-coherence interferometry (a/LCI) is an optical technique that enables depth-specific measurements of nuclear morphology, with applications to detecting epithelial cancers in various organs. Previous a/LCI setups have been limited by costly fiber-optic components and large footprints. Here, we present a novel a/LCI instrument incorporating a channel for optical coherence tomography (OCT) to provide real-time image guidance. We showcase the system's capabilities by acquiring imaging data from in vivo Barrett's esophagus patients. The main innovation in this geometry lies in implementing a pathlength-matched single-mode fiber array, offering substantial cost savings while preserving signal fidelity. A further innovation is the introduction of a specialized side-viewing probe tailored for esophageal imaging, featuring miniature optics housed in a custom 3D-printed enclosure attached to the tip of the endoscope. The integration of OCT guidance enhances the precision of tissue targeting by providing real-time morphology imaging. This novel device represents a significant advancement in clinical translation of an enhanced screening approach for esophageal precancer, paving the way for more effective early-stage detection and intervention strategies.

Esophageal OCT Imaging Using a Paddle Probe Externally Attached to Endoscope.

BACKGROUND AND AIMS: Endoscopic surveillance of Barrett's esophagus (BE) by white light examination is insufficient to diagnose dysplastic change. In this work, we describe an optical imaging method to obtain high-resolution cross-sectional imaging using a paddle-shaped probe affixed to the endoscope tip. METHODS: We integrated Optical Coherence Tomography (OCT), an optical imaging method that produces cross-sectional images, into a paddle probe attached to video endoscope. We acquired images of esophageal epithelium from patients undergoing routine upper GI endoscopy. Images were classified by a reviewer blinded to patient identity and condition, and these results were compared with clinical diagnosis. RESULTS: We successfully captured epithelial OCT images from 30 patients and identified features consistent with both squamous epithelium and Barrett's esophagus. Our blinded image reviewer classified BE versus non-BE with 91.5% accuracy (65/71 image regions), including sensitivity of 84.6% for BE (11/13) and a specificity of 93.1% (54/58). However, in 16 patients, intubation of the probe into the esophagus could not be achieved. CONCLUSIONS: A paddle probe is a feasible imaging format for acquiring cross-sectional OCT images from the esophagus and can provide a structural assessment of BE and non-BE tissue. Probe form factor is the current limiting obstacle, but could be addressed by further miniaturization.

Connectivity-based deep learning approach for segmentation of the epithelium in in vivo human esophageal OCT images.

Optical coherence tomography (OCT) is used for diagnosis of esophageal diseases such as Barrett's esophagus. Given the large volume of OCT data acquired, automated analysis is needed. Here we propose a bilateral connectivity-based neural network for in vivo human esophageal OCT layer segmentation. Our method, connectivity-based CE-Net (Bicon-CE), defines layer segmentation as a combination of pixel connectivity modeling and pixel-wise tissue classification. Bicon-CE outperformed other widely used neural networks and reduced common topological prediction issues in tissues from healthy patients and from patients with Barrett's esophagus. This is the first end-to-end learning method developed for automatic segmentation of the epithelium in in vivo human esophageal OCT images.

Deep imaging with 1.3†µm dual-axis optical coherence tomography and an enhanced depth of focus.

For many clinical applications, such as dermatology, optical coherence tomography (OCT) suffers from limited penetration depth due primarily to the highly scattering nature of biological tissues. Here, we present a novel implementation of dual-axis optical coherence tomography (DA-OCT) that offers improved depth penetration in skin imaging at 1.3 µm compared to conventional OCT. Several unique aspects of DA-OCT are examined here, including the requirements for scattering properties to realize the improvement and the limited depth of focus (DOF) inherent to the technique. To overcome this limitation, our approach uses a tunable lens to coordinate focal plane selection with image acquisition to create an enhanced DOF for DA-OCT. This improvement in penetration depth is quantified experimentally against conventional on-axis OCT using tissue phantoms and mouse skin. The results presented here suggest the potential use of DA-OCT in situations where a high degree of scattering limits depth penetration in OCT imaging.

A review of low-cost and portable optical coherence tomography.

Optical coherence tomography (OCT) is a powerful optical imaging technique capable of visualizing the internal structure of biological tissues at near cellular resolution. For years, OCT has been regarded as the standard of care in ophthalmology, acting as an invaluable tool for the assessment of retinal pathology. However, the costly nature of most current commercial OCT systems has limited its general accessibility, especially in low-resource environments. It is therefore timely to review the development of low-cost OCT systems as a route for applying this technology to population-scale disease screening. Low-cost, portable and easy to use OCT systems will be essential to facilitate widespread use at point of care settings while ensuring that they offer the necessary imaging performances needed for clinical detection of retinal pathology. The development of low-cost OCT also offers the potential to enable application in fields outside ophthalmology by lowering the barrier to entry. In this paper, we review the current development and applications of low-cost, portable and handheld OCT in both translational and research settings. Design and cost-reduction techniques are described for general low-cost OCT systems, including considerations regarding spectrometer-based detection, scanning optics, system control, signal processing, and the role of 3D printing technology. Lastly, a review of clinical applications enabled by low-cost OCT is presented, along with a detailed discussion of current limitations and outlook.

Multimodal Coherent Imaging of Retinal Biomarkers of Alzheimer's Disease in a Mouse Model.

We acquired depth-resolved light scattering measurements from the retinas of triple transgenic Alzheimer's Disease (3xTg-AD) mice and wild type (WT) age-matched controls using co-registered angle-resolved low-coherence interferometry (a/LCI) and optical coherence tomography (OCT). Angle-resolved light scattering measurements were acquired from the nerve fiber layer, outer plexiform layer, and retinal pigmented epithelium using image guidance and segmented thicknesses provided by co-registered OCT B-scans. Analysis of the OCT images showed a statistically significant thinning of the nerve fiber layer in AD mouse retinas compared to WT controls. The a/LCI scattering measurements provided complementary information that distinguishes AD mice by quantitatively characterizing tissue heterogeneity. The AD mouse retinas demonstrated higher mean and variance in nerve fiber layer light scattering intensity compared to WT controls. Further, the difference in tissue heterogeneity was observed through short-range spatial correlations that show greater slopes at all layers of interest for AD mouse retinas compared to WT controls. A greater slope indicates a faster loss of spatial correlation, suggesting a loss of tissue self-similarity characteristic of heterogeneity consistent with AD pathology. Use of this combined modality introduces unique tissue texture characterization to complement development of future AD biomarker analysis.

Reconstruction of angle-resolved backscattering through a multimode fiber for cell nuclei and particle size determination.

We demonstrate reconstruction of angle-resolved optical backscattering after transmission through a multimode fiber. Angle-resolved backscattering is an important tool for particle sizing, and has been developed as a diagnostic modality for detecting epithelial precancer. In this work, we fully characterized the transfer function of a multimode fiber using a plane-wave illumination basis across two dimensions. Once characterized, angle-resolved scattering information which has been scrambled by multimodal propagation can be easily and accurately reconstructed. Our technique was validated using a Mie theory-based inverse light scattering analysis (ILSA) algorithm on polystyrene microsphere phantoms of known sizes. To demonstrate the clinical potential of this approach, nuclear morphology was determined from the reconstructed angular backscattering from MCF-10A human mammary epithelial cell samples and validated against quantitative image analysis (QIA) of fluorescence microscopy images.

First Clinical Application of Low-Cost OCT.

PURPOSE: We present the design of a new low-cost optical coherence tomography (OCT) system and compare its retinal imaging capabilities to a standard commercial system through a clinical study. METHODS: A spectral-domain OCT system was designed using various cost-reduction techniques to be low-cost, highly portable, and completely stand-alone. Clinical imaging was performed on 120 eyes of 60 patients (60 eyes of normal volunteers and 60 eyes with retinal disease) using both the low-cost OCT and a Heidelberg Engineering Spectralis OCT. Contrast-to-noise ratio (CNR) was measured from resulting images to determine system performance. RESULTS: The low-cost OCT system was successfully applied to clinical imaging of the retina. The system offers an axial resolution of 8.0 µm, a lateral resolution of 19.6 µm, and an imaging depth of 2.7 mm for a 6.6-mm field of view in the X and Y directions. Total cost is $5037, a significant size reduction compared to current commercial higher performance systems. Mean CNR value of low-cost OCT images is only 5.6% lower compared to the Heidelberg Spectralis. CONCLUSIONS: The images captured with the low-cost OCT were of adequate resolution and allowed for clinical diagnostics. It offers comparable performance as a retinal screening tool at a fraction of the cost of current commercial systems. TRANSLATIONAL RELEVANCE: Low-cost OCT has the potential to increase access to retinal imaging.

A glycopolymer improves vascoelasticity and mucociliary transport of abnormal cystic fibrosis mucus.

Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.

Origin of improved depth penetration in dual-axis optical coherence tomography: a Monte Carlo study.

Recent studies have demonstrated that extended imaging depth can be achieved using dual-axis optical coherence tomography (DA-OCT). By illuminating and collecting at an oblique angle, multiple forward scattered photons from large probing depths are preferentially detected. However, the mechanism behind the enhancement of imaging depth needs further illumination. Here, the signal of a DA-OCT system is studied using a Monte Carlo simulation. We modeled light transport in tissue and recorded the spatial and angular distribution of photons exiting the tissue surface. Results indicate that the spatial separation and offset angle created by the non-telecentric scanning configuration promote the collection of more deeply propagating photons than conventional on-axis OCT.

Light scattering methods for tissue diagnosis.

Light scattering has become a common biomedical research tool, enabling diagnostic sensitivity to myriad tissue alterations associated with disease. Light-tissue interactions are particularly attractive for diagnostics due to the variety of contrast mechanisms that can be used, including spectral, angle-resolved, and Fourier-domain detection. Photonic diagnostic tools offer further benefit in that they are non-ionizing, non-invasive, and give real-time feedback. In this review, we summarize recent innovations in light scattering technologies, with a focus on clinical achievements over the previous ten years.

Angular range, sampling and noise considerations for inverse light scattering analysis of nuclear morphology.

In recent years, significant work has been devoted to the use of angle-resolved elastic scattering for the extraction of nuclear morphology in tissue. By treating the nucleus as a Mie scattering object, techniques such as angle-resolved low-coherence interferometry (a/LCI) have demonstrated substantial success in identifying nuclear alterations associated with dysplasia. Because optical biopsies are inherently noninvasive, only a small, discretized portion of the 4π scattering field can be collected from tissue, limiting the amount of information available for diagnostic purposes. In this work, we comprehensively characterize the diagnostic impact of variations in angular sampling, range and noise for inverse light scattering analysis of nuclear morphology, using a previously reported dataset from 40 patients undergoing a/LCI optical biopsy for cervical dysplasia. The results from this analysis are applied to a benchtop scanning a/LCI system which compromises angular range for wide-area scanning capability. This work will inform the design of next-generation optical biopsy probes by directing optical design towards parameters which offer the most diagnostic utility.

Real-time speckle reduction in optical coherence tomography using the dual window method.

Speckle is an intrinsic noise of interferometric signals which reduces contrast and degrades the quality of optical coherence tomography (OCT) images. Here, we present a frequency compounding speckle reduction technique using the dual window (DW) method. Using the DW method, speckle noise is reduced without the need to acquire multiple frames. A ~25% improvement in the contrast-to-noise ratio (CNR) was achieved using the DW speckle reduction method with only minimal loss (~17%) in axial resolution. We also demonstrate that real-time speckle reduction can be achieved at a B-scan rate of ~21 frames per second using a graphic processing unit (GPU). The DW speckle reduction technique can work on any existing OCT instrument without further system modification or extra components. This makes it applicable both in real-time imaging systems and during post-processing.

Comparison of imaging fiber bundles for coherence-domain imaging.

Use of imaging fiber bundles for coherence-domain imaging has remained limited to date. In this work, we provide characterization of commercially available imaging bundles for coherence-domain imaging, by evaluating their modal structure for applicability to interferometric imaging. We further examine custom fabricated bundles developed in collaboration with a corporate partner for their ability to reduce interelement optical path length variability and cross talk between elements. The results presented here will serve as a useful guide for comparing fiber bundles for coherence imaging while also offering an improved understanding of the functionality and limitations of imaging bundles for advancing coherent imaging technologies.

Development of an airway mucus defect in the cystic fibrosis rat.

The mechanisms underlying the development and natural progression of the airway mucus defect in cystic fibrosis (CF) remain largely unclear. New animal models of CF, coupled with imaging using micro-optical coherence tomography, can lead to insights regarding these questions. The Cftr-/- (KO) rat allows for longitudinal examination of the development and progression of airway mucus abnormalities. The KO rat exhibits decreased periciliary depth, hyperacidic pH, and increased mucus solid content percentage; however, the transport rates and viscoelastic properties of the mucus are unaffected until the KO rat ages. Airway submucosal gland hypertrophy develops in the KO rat by 6 months of age. Only then does it induce increased mucus viscosity, collapse of the periciliary layer, and delayed mucociliary transport; stimulation of gland secretion potentiates this evolution. These findings could be reversed by bicarbonate repletion but not pH correction without counterion donation. These studies demonstrate that abnormal surface epithelium in CF does not cause delayed mucus transport in the absence of functional gland secretions. Furthermore, abnormal bicarbonate transport represents a specific target for restoring mucus clearance, independent of effects on periciliary collapse. Thus, mature airway secretions are required to manifest the CF defect primed by airway dehydration and bicarbonate deficiency.

Scanning system for angle-resolved low-coherence interferometry.

Angle-resolved low-coherence interferometry (a/LCI) detects precancer by enabling depth-resolved measurements of nuclear morphology in vivo. A significant limitation of a/LCI is the point-probe nature of the method, sampling <0.5 mm2 before probe relocation is necessary. In this work, we demonstrate a scanning method capable of assessing an area >100 mm2 without repositioning. By utilizing a reflection-only three-optic rotator prism and a two-axis scanning mirror, we demonstrate radial scans of a sample with a linear range of 12 mm and a full rotational range of 180°. Use of this design will improve the diagnostic utility of a/LCI for wide-area screening of tissue health.

Neutrophil-Derived Cytosolic PLA2α Contributes to Bacterial-Induced Neutrophil Transepithelial Migration.

Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2, and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of Pseudomonas aeruginosa-induced neutrophil transepithelial migration has not been explored. Human and mouse neutrophil-epithelial cocultures were used to evaluate the role of neutrophil-derived cPLA2α in infection-induced transepithelial signaling by pharmacological and genetic approaches. Primary human airway basal stem cell-derived epithelial cultures and micro-optical coherence tomography, a new imaging modality that captures two- and three-dimensional real-time dynamics of neutrophil transepithelial migration, were applied. Evidence from these studies suggests that cPLA2α expressed by neutrophils, but not epithelial cells, plays a significant role in infection-induced neutrophil transepithelial migration by mediating LTB4 synthesis during migration, which serves to amplify the magnitude of neutrophil recruitment in response to epithelial infection.

Development of a Primary Human Co-Culture Model of Inflamed Airway Mucosa.

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.

Illuminating dynamic neutrophil trans-epithelial migration with micro-optical coherence tomography.

A model of neutrophil migration across epithelia is desirable to interrogate the underlying mechanisms of neutrophilic breach of mucosal barriers. A co-culture system consisting of a polarized mucosal epithelium and human neutrophils can provide a versatile model of trans-epithelial migration in vitro, but observations are typically limited to quantification of migrated neutrophils by myeloperoxidase correlation, a destructive assay that precludes direct longitudinal study. Our laboratory has recently developed a new isotropic 1-µm resolution optical imaging technique termed micro-optical coherence tomography (µOCT) that enables 4D (x,y,z,t) visualization of neutrophils in the co-culture environment. By applying µOCT to the trans-epithelial migration model, we can robustly monitor the spatial distribution as well as the quantity of neutrophils chemotactically crossing the epithelial boundary over time. Here, we demonstrate the imaging and quantitative migration results of our system as applied to neutrophils migrating across intestinal epithelia in response to a chemoattractant. We also demonstrate that perturbation of a key molecular event known to be critical for effective neutrophil trans-epithelial migration (CD18 engagement) substantially impacts this process both qualitatively and quantitatively.