We present a method, based on a single scattering model, to calculate the attenuation coefficient of each pixel in optical coherence tomography (OCT) depth profiles. Numerical simulations were used to determine the model’s response to different depths and attenuation coefficients. Experiments were performed on uniform and layered phantoms with varying attenuation coefficients. They were measured by a 1300 nm OCT system and their attenuation coefficients were evaluated by our proposed method and by fitting the OCT slope as the gold standard. Both methods showed largely consistent results for the uniform phantoms. On the layered phantom, only our proposed method accurately estimated the attenuation coefficients. For all phantoms, the proposed method largely reduced the variability of the estimated attenuation coefficients. The method was illustrated on an in-vivo retinal OCT scan, effectively removing common imaging artifacts such as shadowing. By providing localized, per-pixel attenuation coefficients, this method enables tissue characterization based on attenuation coefficient estimates from OCT data.
(110.3010) Image reconstruction techniques; (110.3200) Inverse scattering; (110.4500) Optical coherence tomography; (170.4500) Optical coherence tomography; (170.4580) Optical diagnostics for medicine; (170.6935) Tissue characterization
We developed an ultrahigh speed, handheld swept source optical coherence tomography (SS-OCT) ophthalmic instrument using a 2D MEMS mirror. A vertical cavity surface-emitting laser (VCSEL) operating at 1060 nm center wavelength yielded a 350 kHz axial scan rate and 10 µm axial resolution in tissue. The long coherence length of the VCSEL enabled a 3.08 mm imaging range with minimal sensitivity roll-off in tissue. Two different designs with identical optical components were tested to evaluate handheld OCT ergonomics. An iris camera aided in alignment of the OCT beam through the pupil and a manual fixation light selected the imaging region on the retina. Volumetric and high definition scans were obtained from 5 undilated normal subjects. Volumetric OCT data was acquired by scanning the 2.4 mm diameter 2D MEMS mirror sinusoidally in the fast direction and linearly in the orthogonal slow direction. A second volumetric sinusoidal scan was obtained in the orthogonal direction and the two volumes were processed with a software algorithm to generate a merged motion-corrected volume. Motion-corrected standard 6 x 6 mm2 and wide field 10 x 10 mm2 volumetric OCT data were generated using two volumetric scans, each obtained in 1.4 seconds. High definition 10 mm and 6 mm B-scans were obtained by averaging and registering 25 B-scans obtained over the same position in 0.57 seconds. One of the advantages of volumetric OCT data is the generation of en face OCT images with arbitrary cross sectional B-scans registered to fundus features. This technology should enable screening applications to identify early retinal disease, before irreversible vision impairment or loss occurs. Handheld OCT technology also promises to enable applications in a wide range of settings outside of the traditional ophthalmology or optometry clinics including pediatrics, intraoperative, primary care, developing countries, and military medicine.
(170.4460) Ophthalmic optics and devices; (170.5755) Retina scanning; (170.3880) Medical and biological imaging; (170.4500) Optical coherence tomography; (170.4470) Ophthalmology
Acquired airway injury is frequently caused by endotracheal intubations, long-term tracheostomies, trauma, airway burns, and some systemic diseases. An effective and less invasive technique for both the early assessment and the early interventional treatment of acquired airway stenosis is therefore needed. Optical coherence tomography (OCT) has been proposed to have unique potential for early monitoring from the proliferative epithelium to the cartilage in acute airway injury. Additionally, stem cell therapy using adipose stem cells is being investigated as an option for early interventional treatment in airway and lung injury. Over the past decade, it has become possible to monitor the level of injury using OCT and to track the engraftment of stem cells using stem cell imaging in regenerative tissue. The purpose of this study was to assess the engraftment of exogenous adipose stem cells in injured tracheal epithelium with fluorescent microscopy and to detect and monitor the degree of airway injury in the same tracheal epithelium with OCT. OCT detected thickening of both the epithelium and basement membrane after tracheal scraping. The engraftment of adipose stem cells was successfully detected by fluorescent staining in the regenerative epithelium of injured tracheas. OCT has the potential to be a high-resolution imaging modality capable of detecting airway injury in combination with stem cell imaging in the same tracheal mucosa.
(170.6935) Tissue characterization; (170.3880) Medical and biological imaging; (170.1610) Clinical applications; (170.4500) Optical coherence tomography
Recent approaches to the analysis of biological samples with three-dimensional linear birefringence orientation require numerical methods to estimate the best fit parameters from experimental measures. We present a novel analytical method for characterizing the intrinsic retardance and the three-dimensional optic axis orientation of uniform and uniaxial turbid media. It is based on a model that exploits the recently proposed differential generalized Jones calculus, remarkably suppressing the need for numerical procedures. The method is applied to the analysis of samples modeled with polarized sensitive Monte Carlo. The results corroborate its capacity to successfully characterize 3D linear birefringence in a straightforward way.
(260.5430) Polarization; (260.2130) Ellipsometry and polarimetry; (110.5405) Polarimetric imaging
Because mid-infrared (mid-IR) spectroscopy is not a promising method to noninvasively measure glucose in vivo, a method for minimally invasive high-precision glucose determination in vivo by mid-IR laser spectroscopy combined with a tunable laser source and small fiber-optic attenuated total reflection (ATR) sensor is introduced. The potential of this method was evaluated in vitro. This research presents a mid-infrared tunable laser with a broad emission spectrum band of 9.19 to 9.77μm(1024~1088 cm−1) and proposes a method to control and stabilize the laser emission wavelength and power. Moreover, several fiber-optic ATR sensors were fabricated and investigated to determine glucose in combination with the tunable laser source, and the effective sensing optical length of these sensors was determined for the first time. In addition, the sensitivity of this system was four times that of a Fourier transform infrared (FT-IR) spectrometer. The noise-equivalent concentration (NEC) of this laser measurement system was as low as 3.8 mg/dL, which is among the most precise glucose measurements using mid-infrared spectroscopy. Furthermore, a partial least-squares regression and Clarke error grid were used to quantify the predictability and evaluate the prediction accuracy of glucose concentration in the range of 5 to 500 mg/dL (physiologically relevant range: 30~400 mg/dL). The experimental results were clinically acceptable. The high sensitivity, tunable laser source, low NEC and small fiber-optic ATR sensor demonstrate an encouraging step in the work towards precisely monitoring glucose levels in vivo.
(170.1470) Blood or tissue constituent monitoring; (300.1030) Absorption; (300.6340) Spectroscopy, infrared
Intravascular optical frequency-domain imaging (OFDI), a second-generation optical coherence tomography (OCT) technology, enables imaging of the three-dimensional (3D) microstructure of the vessel wall following a short and nonocclusive clear liquid flush. Although 3D vascular visualization provides a greater appreciation of the vessel wall and intraluminal structures, a longitudinal imaging pitch that is several times bigger than the optical imaging resolution of the system has limited true high-resolution 3D imaging, mainly due to the slow scanning speed of previous imaging catheters. Here, we demonstrate high frame-rate intravascular OFDI in vivo, acquiring images at a rate of 350 frames per second. A custom-built, high-speed, and high-precision fiber-optic rotary junction provided uniform and high-speed beam scanning through a custom-made imaging catheter with an outer diameter of 0.87 mm. A 47-mm-long rabbit aorta was imaged in 3.7 seconds after a short contrast agent flush. The longitudinal imaging pitch was 34 μm, comparable to the transverse imaging resolution of the system. Three-dimensional volume-rendering showed greatly enhanced visualization of tissue microstructure and stent struts relative to what is provided by conventional intravascular imaging speeds.
(170.4500) Optical coherence tomography; (170.2150) Endoscopic imaging; (170.3880) Medical and biological imaging
The collagen meshwork plays a central role in the functioning of a range of tissues including cartilage, tendon, arteries, skin, bone and ligament. Because of its importance in function, it is of considerable interest for studying development, disease and regeneration processes. Here, we have used second harmonic generation (SHG) to image human tissues on the hundreds of micron scale, and developed a numerical model to quantitatively interpret the images in terms of the underlying collagen structure on the tens to hundreds of nanometer scale. Focusing on osteoarthritic changes in cartilage, we have demonstrated that this combination of polarized SHG imaging and numerical modeling can estimate fibril diameter, filling fraction, orientation and bundling. This extends SHG microscopy from a qualitative to quantitative imaging technique, providing a label-free and non-destructive platform for characterizing the extracellular matrix that can expand our understanding of the structural mechanisms in disease.
(170.0170) Medical optics and biotechnology; (190.0190) Nonlinear optics
Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution.
(180.2520) Fluorescence microscopy; (100.0100) Image processing; (100.3010) Image reconstruction techniques; (100.6640) Superresolution
Three-dimensional imaging of the mucosa of the lower lip and labial minor salivary glands is demonstrated in vivo using swept source optical coherence tomography (OCT) system at 1310 nm with modified interface. Volumetric data sets of the inner surface of the lower lip covering ~230 mm2 field are obtained from patients with Sjögren’s syndrome and a control group. OCT enables high-resolution visualization of mucosal architecture using cross-sectional images as well as en-face projection images. Comprehensive morphometry of the labial minor salivary glands is performed, and statistical significance is assessed. Statistically significant differences in morphometric parameters are found when subgroups of patients with Sjögren’s syndrome are analyzed.
(110.4500) Optical coherence tomography; (170.3880) Medical and biological imaging; (170.4580) Optical diagnostics for medicine; (170.6930) Tissue
As part of a proof-of-concept study for future delivery of targeted near-infrared fluorescent (NIRF) tracers, we sought to assess the delivery of micrograms of indocyanine green to all the axillary lymph nodes following intraparenchymal breast injections and intradermal arm injections in 20 subjects with advanced breast carcinoma and undergoing complete axillary lymph node dissection. Lymphatic vessels and nodes were assessed in vivo. Ex vivo images demonstrated that 87% of excised lymph nodes, including 81% of tumor-positive lymph nodes, were fluorescent. Future clinical studies using microdose amounts of tumor-targeting NIRF contrast agents may demonstrate improved surgical intervention with reduced morbidity.
(100.2960) Image analysis; (170.0110) Imaging systems; (170.1610) Clinical applications; (170.3880) Medical and biological imaging
Eosinophilic esophagitis (EoE) is an allergic condition that is characterized by eosinophils infiltrating the esophageal wall. The treatment of the disease may require multiple follow up sedated endoscopies and biopsies to confirm elimination of eosinophils. These procedures are expensive, time consuming, and may be difficult for patients to tolerate. Here we report on the development of a confocal microscopy capsule for diagnosis and monitoring of EoE. The swallowable capsule implements a high-speed fiber-based reflectance confocal microscopy technique termed Spectrally Encoded Confocal Microscopy (SECM). SECM scans the sample in one dimension without moving parts by using wavelength swept source illumination and a diffraction grating at the back plane of the objective lens. As the wavelength of the source is tuned, the SECM optics within the 7 x 30 mm capsule are rotated using a driveshaft enclosed in a 0.8 mm flexible tether. A single rotation of the optics covered a field of view of 22 mm x 223 µm. The lateral and axial resolutions of the device were measured to be 2.1 and 14 µm, respectively. Images of Acetic Acid stained swine esophagus obtained with the capsule ex vivo and in vivo clearly showed squamous epithelial nuclei, which are smaller and less reflective than eosinophils. Imaging of esophageal biopsies from EoE patients ex vivo demonstrated the capability of this technology to visualize individual eosinophils. Based on the results of this study, we believe that this capsule will be a simpler and more effective device for diagnosing EoE and monitoring the therapeutic response of this disease.
(170.1790) Confocal microscopy; (170.2150) Endoscopic imaging; (170.2680) Gastrointestinal; (170.4730) Optical pathology
A 3D living-cell culture in hydrogel has been developed as a standardized low-tensile-strength tissue proxy for study of ultrafast, pulsetrain-burst laser-tissue interactions. The hydrogel is permeable to fluorescent biomarkers and optically transparent, allowing viable and necrotic cells to be imaged in 3D by confocal microscopy. Good cell-viability allowed us to distinguish between typical cell mortality and delayed subcellular tissue damage (e.g., apoptosis and DNA repair complex formation), caused by laser irradiation. The range of necrosis depended on laser intensity, but not on pulsetrain-burst duration. DNA double-strand breaks were quantified, giving a preliminary upper limit for genetic damage following laser treatment.
(170.1020) Ablation of tissue; (350.3390) Laser materials processing; (320.7090) Ultrafast lasers; (140.3440) Laser-induced breakdown; (160.1435) Biomaterials; (170.2520) Fluorescence microscopy
We have developed an extremely miniaturized optical coherence tomography (OCT) needle probe (outer diameter 310 µm) with high sensitivity (108 dB) to enable minimally invasive imaging of cellular structure deep within skeletal muscle. Three-dimensional volumetric images were acquired from ex vivo mouse tissue, examining both healthy and pathological dystrophic muscle. Individual myofibers were visualized as striations in the images. Degradation of cellular structure in necrotic regions was seen as a loss of these striations. Tendon and connective tissue were also visualized. The observed structures were validated against co-registered hematoxylin and eosin (H&E) histology sections. These images of internal cellular structure of skeletal muscle acquired with an OCT needle probe demonstrate the potential of this technique to visualize structure at the microscopic level deep in biological tissue in situ.
(060.2370) Fiber optics sensors; (170.4500) Optical coherence tomography; (170.6935) Tissue characterization; (230.3990) Micro-optical devices
Tissue Engineering is a new emerging field that offers many possibilities to produce three-dimensional and functional tissues like ligaments or scaffolds. The biocompatibility of these materials is crucial in tissue engineering, since they should be integrated in situ and should induce a good cell adhesion and proliferation. One of the most promising materials used for tissue engineering are polyesters such as Poly-ε-caprolactone (PCL), which is used in this work. In our case, the bio-integration is reached by grafting a bioactive polymer (pNaSS) on a PCL surface. Using nonlinear microscopy, PCL structure is visualized by SHG and proteins and cells by two-photon excitation autofluorescence generation. A comparative study between grafted and nongrafted polymer films is provided. We demonstrate that the polymer grafting improves the protein adsorption by a factor of 75% and increase the cell spreading onto the polymer surface. Since the spreading is directly related to cell adhesion and proliferation, we demonstrate that the pNaSS grafting promotes PCL biocompatibility.
(160.1435) Biomaterials; (180.4315) Nonlinear microscopy
We developed a label-free nonlinear optical (NLO) microscope integrating the stimulated Raman scattering, multi-color two-photon excited fluorescence and second harmonic generation. The system produces multimodal images of protein content, mitochondria distribution and sarcomere structure of fresh muscle samples. With the advanced imaging technique, we studied the mal-development of skeletal muscle caused by sarcomeric gene deficiency. In addition, important development processes of normal muscle from neonatal to adult stage were also clearly revealed based on the changing sarcomere structure, mitochondria distribution and muscle fiber size. The results demonstrate that the newly developed multimodal NLO microscope is a powerful tool to assess the muscle integrity and function.
(170.3880) Medical and biological imaging; (170.6930) Tissue; (180.4315) Nonlinear microscopy; (290.5910) Scattering, stimulated Raman
Dual-band Fourier domain optical coherence tomography (FD-OCT) provides depth-resolved spectroscopic imaging that enhances tissue contrast and reduces image speckle. However, previous dual-band FD-OCT systems could not correctly give the tissue spectroscopic contrast due to depth-related discrepancy in the imaging method and attenuation in biological tissue samples. We designed a new dual-band full-range FD-OCT imaging system and developed an algorithm to compensate depth-related fall-off and light attenuation. In our imaging system, the images from two wavelength bands were intrinsically overlapped and their intensities were balanced. The processing time of dual-band OCT image reconstruction and depth-related compensations were minimized by using multiple threads that execute in parallel. Using the newly developed system, we studied tissue phantoms and human cancer xenografts and muscle tissues dissected from severely compromised immune deficient mice. Improved spectroscopic contrast and sensitivity were achieved, benefiting from the depth-related compensations.
(110.4500) Optical coherence tomography; (170.3880) Medical and biological imaging
We present a novel polarization sensitive optical coherence tomography (PS-OCT) system with an integrated retinal tracker. The tracking operates at up to 60 Hz, correcting PS-OCT scanning positions during the acquisition to avoid artifacts caused by eye motion. To demonstrate the practical performance of the system, we imaged several healthy volunteers and patients with AMD both with B-scan repetitions for frame averaging and with 3D raster scans. Under large retinal motions with up to 1 mm amplitude at 0.5 ~a few Hz frequency range, motion artifact suppression in the PS-OCT images as well as standard deviation noise reduction in the frame averaged retardation images are presented.
(170.0170) Medical optics and biotechnology; (170.0110) Imaging systems; (170.2655) Functional monitoring and imaging; (170.4470) Ophthalmology; (170.4500) Optical coherence tomography
This work presents, to our knowledge, the first demonstration of the Laser Speckle Contrast
Imaging (LSCI) technique with extended depth of field (DOF). We employ wavefront coding on the
detected beam to gain quantitative information on flow speeds through a DOF extended two-fold
compared to the traditional system. We characterize the system in-vitro using
controlled microfluidic experiments, and apply it in-vivo to imaging the
somatosensory cortex of a rat, showing improved ability to image flow in a larger number of vessels
(110.6150) Speckle imaging; (110.7348) Wavefront encoding; (170.6480) Spectroscopy, speckle
Cardiovascular disease typically is associated with dysfunction of the coronary vasculature and microvasculature. The study of cardiovascular disease typically involves imaging of the large coronary vessels and quantification of cardiac blood perfusion. These methods, however, are not well suited for imaging of the cardiac microvasculature. We used the optical histology method, which combines chemical optical clearing and optical imaging, to create high-resolution, wide-field maps of the cardiac microvasculature in ventral slices of mouse heart. We have demonstrated the ability of the optical histology method to enable wide-field visualization of the cardiac microvasculature in high-resolution and anticipate that optical histology may have significant impact in studying cardiovascular disease.
(170.0170) Medical optics and biotechnology; (170.3880) Medical and biological imaging; (170.6930) Tissue; (170.1790) Confocal microscopy; (000.1430) Biology and medicine
White-light surveillance colonoscopy is the standard of care for the detection and removal of premalignant lesions to prevent colorectal cancer, and the main screening recommendation following treatment for recurrence detection. However, it lacks sufficient diagnostic yield, exhibits unacceptable adenoma miss-rates and is not capable of revealing functional and morphological information of the detected lesions. Fluorescence molecular guidance in the near-infrared (NIR) is expected to have outstanding relevance regarding early lesion detection and heterogeneity characterization within and among lesions in these interventional procedures. Thereby, superficial and sub-surface tissue biomarkers can be optimally visualized due to a minimization of tissue attenuation and autofluorescence by comparison with the visible, which simultaneously enhance tissue penetration and assure minimal background. At present, this potential is challenged by the difficulty associated with the clinical propagation of disease-specific contrast agents and the absence of a commercially available endoscope that is capable of acquiring wide-field, NIR fluorescence at video-rates. We propose two alternative flexible endoscopic fluorescence imaging methods, each based on a CE certified commercial, clinical grade endoscope, and the employment of an approved monoclonal antibody labeled with a clinically applicable NIR fluorophore. Pre-clinical validation of these two strategies that aim at bridging NIR fluorescence molecular guidance to clinical translation is demonstrated in this study.
(170.2150) Endoscopic imaging; (170.2680) Gastrointestinal
A simple method of simulating possible coded aperture phase contrast X-ray imaging apparatus is presented. The method is based on ray tracing, with the rays treated ballistically within a voxelized sample and with the phase-shift-induced angular deviations and absorptions applied at a plane in the middle of the sample. For the particular case of a coded aperture phase contrast configuration suitable for small animal pre-clinical imaging we present results obtained using a high resolution voxel array representation of a mathematically-defined ‘digital’ mouse. At the end of the article a link to the software is supplied.
(340.7440) X-ray imaging; (170.3880) Medical and biological imaging
The capability to perform high-resolution, wide field-of-view (FOV) microscopy imaging is highly sought after in biomedical applications. In this paper, we report a wide FOV microscopy system that uses a closed-circuit-television (CCTV) lens for image relay and a flatbed scanner for data acquisition. We show that such an imaging system is capable of capturing a 10 mm × 7.5 mm FOV image with 0.78 µm resolution, resulting in more than 0.5 billion pixels across the entire image. The resolution and field curve of the proposed system were characterized by imaging a USAF resolution target and a hole-array target. To demonstrate its application, 0.5 gigapixel images of histology slides were acquired using this system.
(120.4820) Optical systems; (170.0180) Microscopy; (170.4730) Optical pathology
Proof of concept results are presented towards an in situ bimodal proximity sensor for neurovascular bundle detection during dental implant surgery using combined near infrared absorption (NIR) and optical coherence tomography (OCT) techniques. These modalities are shown to have different sensitivity to the proximity of optical contrast from neurovascular bundles. NIR AC and DC signals from the pulsing of an artery enable qualitative ranging of the bundle in the millimeter range, with best sensitivity around 0.5-3mm distance in a custom phantom setup. OCT provides structural mapping of the neurovascular bundle at sub-millimeter distances in an ex vivo human jaw bone. Combining the two techniques suggests a novel ranging system for the surgeon that could be implemented in a “smart drill.” The proximity to the neurovascular bundle can be tracked in real time in the range of a few millimeters with NIR signals, after which higher resolution imaging OCT to provide finer ranging in the sub-millimeter distances.
(170.0170) Medical optics and biotechnology; (170.1850) Dentistry; (170.1610) Clinical applications; (170.4500) Optical coherence tomography; (170.6510) Spectroscopy, tissue diagnostics
Lens average and equivalent refractive indices are required for purposes such as lens thickness estimation and optical modeling. We modeled the refractive index gradient as a power function of the normalized distance from lens center. Average index along the lens axis was estimated by integration. Equivalent index was estimated by raytracing through a model eye to establish ocular refraction, and then backward raytracing to determine the constant refractive index yielding the same refraction. Assuming center and edge indices remained constant with age, at 1.415 and 1.37 respectively, average axial refractive index increased (1.408 to 1.411) and equivalent index decreased (1.425 to 1.420) with age increase from 20 to 70 years. These values agree well with experimental estimates based on different techniques, although the latter show considerable scatter. The simple model of index gradient gives reasonable estimates of average and equivalent lens indices, although refinements in modeling and measurements are required.
(330.4460) Ophthalmic optics and devices; (330.7326) Visual optics, modeling
A two-layer Monte Carlo lookup table-based inverse model is validated with two-layered phantoms across physiologically relevant optical property ranges. Reflectance data for source-detector separations of 370 μm and 740 μm were collected from these two-layered phantoms and top layer thickness, reduced scattering coefficient and the top and bottom layer absorption coefficients were extracted using the inverse model and compared to the known values. The results of the phantom verification show that this method is able to accurately extract top layer thickness and scattering when the top layer thickness ranges from 0 to 550 μm. In this range, top layer thicknesses were measured with an average error of 10% and the reduced scattering coefficient was measured with an average error of 15%. The accuracy of top and bottom layer absorption coefficient measurements was found to be highly dependent on top layer thickness, which agrees with physical expectation; however, within appropriate thickness ranges, the error for absorption properties varies from 12–25%.
(170.6510) Spectroscopy, tissue diagnostics; (100.3190) Inverse problems