We present a dual-modality instrument that utilizes a paddle-shaped probe to conduct OCT imaging and a/LCI nuclear size measurements simultaneously to provide both structural and functional information in a single device during an endoscopic procedure. The probe is 3D printed using biocompatible material to deliver both modalities to the same location on the esophageal epithelium as a means to detect pre-cancerous tissues. We will present an update of our current clinical study at the University of North Carolina Center for Esophageal Diseases and Swallowing (UNC CEDAS) with a target of 40 total patients with and without esophageal dysplasia.
We present a method for large volume imaging of highly scattering tissues using a dual-axis optical coherence tomography system (DA-OCT) at 1.3 μm featuring a dynamic focus-tracking method to create an enhanced depth of focus. Our approach is validated for skin imaging, using an in vivo rat skin model. A quantitative discussion of imaging performance in highly scattering tissue for both DA-OCT and conventional OCT at 1.3 μm is presented.
We present results from depth-resolved light scattering measurements of triple transgenic mouse retinas for Alzheimer’s Disease (AD) using a multimodal coherent imaging system. Use of a co-registered angle-resolved low-coherence interferometry (a/LCI) and optical coherence tomography (OCT) system allows unique analysis that is otherwise unavailable using a single modality to provide complementary information on tissue structural changes associated with AD. This abstract summarizes the light scattering parameters drawn using this system at selective retinal layers guided by OCT image segmentation. Future developments of this combined system for human retinal imaging, which involve a low-cost OCT engine, are also discussed.
Angle-resolved low-coherence interferometry (a/LCI) is an optical technique which uses inverse models of light scattering to predict the size and density of cell nuclei, a significant biomarker of precancer. In recent years, substantial progress has occurred in a/LCI technology, particularly in novel instrumentation for the detection of dysplasia in the cervix and the esophagus. This abstract summarizes recent developments in a/LCI, with a focus on developments over the previous four years. Clinical studies in the cervix, novel optical instrumentation addressing the cervix and esophagus, and novel applications related to Alzheimer’s disease are discussed.
Video endoscopy remains the most common means of examining the upper gastrointestinal tract. However, white-light widefield imagery is limited in diagnostic sensitivity and specificity for conditions such as Barrett’s esophagus. To provide cross-sectional imaging that yields more accurate diagnosis, optical coherence tomography (OCT) has been implemented for upper GI screening and diagnosis in several form factors, such as inflatable balloons and tethered capsules.
We now present an alternative configuration for esophageal OCT. Our OCT probe is contained within an articulating paddle that attaches to the end of an upper GI endoscope via flexible cuff. This arrangement allows regions of interest to be visually identified by the physician operating the endoscope and targeted for OCT imaging by application of the paddle.
The probe housing was 3D printed using biocompatible dental resin. The flexible cuff was also 3D printed using a silicone-based resin to allow a tight fit to the endoscope. The optical probe was a rotating fiber-optic design, consisting of a gradient-index lens and prism on the distal end, wound steel torque coil, a polymer sheath, and a fiber-optic rotary junction. The OCT system was a spectral domain configuration with custom spectrometer operating at 20,000 A-lines per second. The illumination source was a superluminescent diode centered at 1310 nm.
A clinical pilot study at the University of North Carolina Endoscopy Center is scheduled to begin imminently.
Optical imaging techniques generally offer shallow penetration depths due to high scattering in biological tissue. We have recently developed dual-axis optical coherence tomography (DA-OCT) for interferometric imaging at extended depths. By illuminating and collecting at oblique angles, multiply forward scattered photons from deeper penetration depths were favorably detected. A Monte Carlo study demonstrated a >12-fold increase in signal-to-background ratio (SBR) in skin tissue at 1 mm depth by DA-OCT. A 1300 nm DA-OCT system was designed and constructed, offering an imaging depth of up to 2 mm in skin tissue
The early detection of cervical dysplasia enables early treatment, a critical factor in cancer prevention. In the United States, cervical cancer screening is age-based and includes cervical cytology with human papilloma virus (HPV) testing with referral to colposcopy for abnormal results. Colposcopy is used to visualize changes in the appearance of the transformation zone to direct biopsies which can confirm a diagnosis of dysplasia or cancer. Directed biopsies can be limited in detection of abnormalities because they represent a small area of the transformation zone and can be limited by provider expertise. Additionally, biopsies contribute to patient discomfort and anxiety awaiting for results.
We recently reported the first in vivo cervical data from angle-resolved low-coherence interferometry (a/LCI), an optical technique that measures nuclear size as a biomarker for dysplasia, which is well-suited for screening due to its high sensitivity and specificity and its non-invasive utilization. However, in order to target the single-point measurements of the a/LCI instrument, we aimed to construct a probe capable of mapping the cervical epithelium to identify the transformation zone between the ectocervical and endocervical epithelia, the location at which dysplasia is most likely to develop.
We termed this complementary technology multiplexed low-coherence interferometer (m/LCI). Thirty-six parallel fiber-optic interferometers were constructed to obtain optical depth profiles using spectral-domain LCI. Light from each channel is delivered to the cervix via a 6x6 fiber-optic bundle and a custom endoscopic probe. The depth-profile from each optical channel enables the identification of the ectocervix and endocervix.
A pilot study at Duke University (n=5) was followed by an ongoing clinical study at New York City Health + Hospitals/Jacobi (Bronx, New York) (current n=20, target n=50). We present the results from these first studies to demonstrate the feasibility of m/LCI as a means of identifying the transformation zone for screening of dysplasia.
The intestinal epithelial barrier provides protection from external threats that enter the digestive system and persist beyond passage through the stomach. The effects of toxic agents on the intestinal epithelial cell monolayer have not been fully characterized at a cellular level as live imaging of this dynamic interplay at sufficient resolution to interpret cellular responses presents technological challenges. Using a high-resolution native contrast modality called Micro-Optical Coherence Tomography (μOCT), we generated real-time 3D images depicting the impact of the chemical agent EDTA on polarized intestinal epithelial monolayers. Within minutes following application of EDTA, we observed a change in the uniformity of epithelial surface thickness and loss of the edge brightness associated with the apical surface. These observations were measured by generating computer algorithms which quantify imaged-based events changing over time, thus providing parallel graphed data to pair with video. The imaging platform was designed to monitor epithelial monolayers prior to and following application of chemical agents in order to provide a comprehensive account of monolayer behavior at baseline conditions and immediately following exposure. Furthermore, the platform was designed to simultaneously measure continuous trans-epithelial electric resistance (TEER) in order to define the progressive loss of barrier integrity of the cell monolayer following exposure to toxic agents and correlate these findings to image-based metrics. This technological image-based experimental platform provides a novel means to characterize mechanisms that impact the intestinal barrier and, in future efforts, can be applied to study the impact of disease relevant agents such as enteric pathogens and enterotoxins.
The cervix is primarily composed of two types of epithelium: stratified squamous ectocervix and simple columnar endocervix. In between these two layers lies a metaplastic squamocolumnar junction commonly referred to as the transformation zone (T-zone). During puberty, the cervical epithelium undergoes dynamic changes including cervical ectropion and increased area and rates of metaplasia. Although these metaplastic changes have been linked to higher incidence of cervical cancer among young women, research in this field has been limited to surface analysis using computerized planimetry of colopophotographs.
Here, we present a novel multiplexed low coherence interferometry (mLCI) system for interrogating the cervical epithelium. The system is comprised of 6 parallel Mach-Zehnder interferometers in a time-multiplexed configuration that increases throughput by 6-fold to realize a combined 36-channel acquisition. A custom designed endoscopic handheld probe is used to collect sparsely sampled, depth-resolved scattering intensity profiles (A-scans) from a large field of view (25 x 25 mm) on the cervical epithelium in vivo. The instrument incorporates white light imaging through a plastic fiber bundle to co-register the mLCI A-scans to colpophotographs which are analyzed by a clinician to manually segment the cervical epithelium. Our preliminary data shows significant differences in characteristic A-scans from endocervical and ectocervical epithelium. These results demonstrate the feasibility of using mLCI as both a research tool for studying the relationship between cervical ectopy and cancer as well as a clinical instrument for identifying the at-risk T-zone on the cervix in vivo as a means to improve biopsy targeting. Further analysis will be performed to develop an algorithm for distinguishing the mLCI A-scans of endocervical, ectocervical, and metaplastic epithelium in real time.
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.
KEYWORDS: Tomography, Coherence (optics), Signal detection, Visualization, 3D image processing, Receptors, Resistance, Biomedical optics, Current controlled current source
Neutrophils are immune cells that undergo chemotaxis, detecting and migrating towards a chemical signal gradient. Neutrophils actively migrate across epithelial boundaries, interacting with the epithelium to selectively permit passage without compromising the epithelial barrier. In many inflammatory disorders, excessive neutrophil migration can cause damage to the epithelium itself. The signaling pathways and mechanisms that facilitate trans-epithelial migration are not fully characterized. Our laboratory has developed micro-optical coherence tomography (μOCT), which has 2 μm lateral resolution and 1 μm axial resolution. As a high-resolution native contrast modality, μOCT can directly visualize individual neutrophils as they interact with a cell layer cultured on a transwell filter. A chemoattractant can be applied to the apical side of inverted monolayer, and human neutrophils placed in the basolateral compartment, while μOCT captures 3D images of the chemotaxis. μOCT images can also generate quantitative metrics of migration volume to study the dependence of chemotaxis on monolayer cell type, chemoattractant type, and disease state of the neutrophils. For example, a disease known as leukocyte adhesion deficiency (LAD) can be simulated by treating neutrophils with antibodies that interfere with the CD18 receptor, a facilitator of trans-epithelial migration. We conducted a migration study of anti-CD18 treated and control neutrophils using T84 intestinal epithelium as a barrier. After one hour, μOCT time-lapse imaging indicated a strong difference in the fraction of neutrophils that remain attached to the epithelium after migration (0.67 ± 0.12 attached anti-CD18 neutrophils, 0.23 ± 0.08 attached control neutrophils, n = 6, p < 0.05), as well as a modest but non-significant decrease in total migration volume for treated neutrophils. We can now integrate μOCT-derived migration metrics with simultaneously acquired measurements of transepithelial electrical resistance (TEER), a measure of membrane integrity that decreases when neutrophils create openings in the epithelium to permit migration. Preliminary results (n = 2) using real-time TEER measurements indicate that TEER change in anti-CD18 migration (26% at 1 hour) is not lower compared to control (14% at 1 hour), suggesting that the neutrophil-epithelial interaction is not impaired. Combined µOCT+TEER will allow the relationship of neutrophil migration and epithelial interactions to be studied to help uncover the mechanisms of altered neutrophil behavior in patients with inflammatory and immune diseases.
Mucociliary clearance (MCC) plays a significant role in maintaining the health of human respiratory system by eliminating foreign particles trapped within mucus. Failure of this mechanism in diseases such as cystic fibrosis and chronic obstructive pulmonary disease (COPD) leads to airway blockage and lung infection, causing morbidity and mortality. The volume of airway mucus and the periciliary liquid encapsulating the cilia, in addition to ciliary beat frequency and velocity of mucociliary transport, are vital parameters of airway health. However, the diagnosis of disease pathogenesis and advances of novel therapeutics are hindered by the lack of tools for visualization of ciliary function in vivo.
Our laboratory has previously developed a 1-µm resolution optical coherence tomography method, termed Micro-OCT, which is capable of visualizing mucociliary transport and quantitatively capturing epithelial functional metrics. We have also miniaturized Micro-OCT optics in a first-generation rigid 4mm Micro-OCT endoscope utilizing a common-path design and an apodizing prism configuration to produce an annular profile sample beam, and reported the first in vivo visualization of mucociliary transport in swine. We now demonstrate a flexible 2.5 mm Micro-OCT probe that can be inserted through the instrument channel of standard flexible bronchoscopes, allowing bronchoscopic navigation to smaller airways and greatly improving clinical utility. Longitudinal scanning over a field of view of more than 400 µm at a frame rate of 40 Hz was accomplished with a driveshaft transduced by a piezo-electric stack motor.
We present characterization and imaging results from the flexible micro-OCT probe and progress towards clinical translation. The ability of the bronchoscope-compatible micro-OCT probe to image mucus clearance and epithelial function will enable studies of cystic fibrosis pathogenesis in small airways, provide diagnosis of mucociliary clearance disorders, and allow individual responses to treatments to be monitored.
We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.
We present an endomicroscope apparatus that exhibits out-of-focus background rejection based on wide-field illumination through a flexible imaging fiber bundle. Our technique, called HiLo microscopy, involves acquiring two images, one with grid-pattern illumination and another with standard uniform illumination. An evaluation of the image contrast with grid-pattern illumination provides an optically sectioned image with low resolution. This is complemented with high-resolution information from the uniform illumination image, leading to a full-resolution image that is optically sectioned. HiLo endomicroscope movies are presented of fluorescently labeled rat colonic mucosa.
We report our progress in the development of Differential Aberration Imaging (DAI), a technique that enhances twophoton
fluorescence (TPEF) microscopy by improving rejection of out-of-focus background by means of a deformable
mirror (DM). The DM is used to intentionally add aberrations to the imaging system, which causes dramatic losses to
in-focus signal while preserving the bulk of the out-of-focus background. By taking the difference between TPEF
images with and without added aberrations, the out-of-focus portion of the signal is further rejected. We now introduce
an implementation of DAI using a new type of DM that can be produced at much lower cost.
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