We have developed a multispectral imaging technique for identifying chemical compounds in vivo in the retina with cellular-level resolution and without the use of contrast agents. We combined simultaneous multi-channels offset and confocal AO-SLO imaging, which provides isotropic images of retinal microstructures free of directionality artifacts, with spectral signature analysis of chemical compounds to identify such biomarkers at cellular-level. The new concept has been demonstrated in a model eye using commercially available Aβ. An animal study on a mouse Alzheimer’s model is ongoing. This technique may pave a path forward for better understanding of the onset of various neurodegenerative diseases.
Simultaneous dual-channel offset imaging provides isotropic images of retinal microstructures that enable phase imaging in the living eye with enhanced visualization contrast. Phase imaging is widely used in microscopy and biomedical imaging to reveal structures not visible in standard imaging due to their low scattering properties. We implement the technique in a line-scanning approach using a high-speed 2D camera to visualize microstructures in the living eye with enhanced contrast, that were only visible with other modalities, such as flying-spot scanning laser ophthalmoscopy (SLO). A simplified phase imaging system has the potential to be quantitative, with diagnostic value for retinal diseases, and may enable monitoring treatment. Methods for super-resolution reconstruction were explored to break the diffraction limit. SLO phase imaging exploits forward-scatter through phase objects in the retina and subsequent reflection (rescatter) of intensity-encoded diffuse reflections for detection; line-scan ophthalmoscopy (LSO) phase imaging works in the opposite way, in which the offset line-beam produces oblique back-illumination enabling the diffractive/refractive effects of phase objects in the inner retina to be imaged in transmission. The former scrambles optical phase information, the latter preserves it. This design has several advantages over conventional SLOs: 1) the LSO has a reduced number of optical elements, which results in a short optical path and compact design, 2) only one moving part, thus hardware and electronics are simplified, and 3) the LSO is inherently safer because the beam is focused in only one dimension on the retina.
Simultaneous multi-channels offset imaging provides isotropic images of retinal microstructures free of directionality artifacts and enables phase imaging in the living eye with enhanced visualization contrast. Phase imaging is widely used in microscopy and biomedical imaging to reveal structures not visible in standard imaging due to their low scattering properties. We introduce the technique in high-resolution retinal imaging to visualize microstructures in the living eye with enhanced contrast, that were not visible with other modalities, and without adding contrast agents. Phase imaging has the potential to be quantitative, may have diagnostic value for retinal diseases, and may enable monitoring treatment.
In this paper we report the use of a novel multimodal imaging hand-held probe for guiding laser and radiotherapy on nonmelanoma skin cancers (NMSCs) patients. This probe combines the capability of reflectance confocal microscopy (RCM) with that of Optical Coherence Tomography (OCT) to reliably detect cancer markers and measure cancer depth of invasion. These capabilities have shown to be very effective in accurately measuring cancer margins and guiding the therapy.
We developed a novel ophthalmic imaging platform that combines non-invasive measurements of retina/choroid structure and ocular blood flow based on optical coherence tomography (OCT) and wide-field semi-quantitative global flow visualization using line-scanning Doppler flowmetry (LSDF). The combination of these two imaging modalities within the same imaging platform enables comprehensive assessment of blood flow in retina and choroid and provides efficient characterization of blood flow in hemodynamic studies both in human volunteers and in small animals. The platform enables visualization of the entire posterior hemisphere vasculature, including vortex veins, using only light and without additional contrast agent in humans and rats.
A new detection scheme was developed for simultaneous multi-channel imaging that provides isotropic images of retinal structures, free of directionality artifacts. The arrangement consists of light collecting fibers that act as offset apertures. This fiber bundle configuration can be used to retrofit basically any existing AO-SLO platform. The channels can be combined to reveal additional structural and functional details and this kind of retinal imaging with cellular resolution is a valuable new tool for researchers and clinicians.
The use of a Prosthetic Replacement of the Ocular Surface Environment (PROSE) device is a revolutionary treatment for military patients that have lost their eyelids due to 3rd degree facial burns and for civilians who suffer from a host of corneal diseases. However, custom manual fitting is often a protracted painful, inexact process that requires multiple fitting sessions. Training for new practitioners is a long process. Automated methods to measure the complete corneal and scleral topology would provide a valuable tool for both clinicians and PROSE device manufacturers and would help streamline the fitting process.
PSI has developed an ocular anterior-segment profiler based on Optical Coherence Tomography (OCT), which provides a 3D measure of the surface of the sclera and cornea. This device will provide topography data that will be used to expedite and improve the fabrication process for PROSE devices. OCT has been used to image portions of the cornea and sclera and to measure surface topology for smaller contact lenses [1-3]. However, current state-of-the-art anterior eye OCT systems can only scan about 16 mm of the eye’s anterior surface, which is not sufficient for covering the sclera around the cornea. In addition, there is no systematic method for scanning and aligning/stitching the full scleral/corneal surface and commercial segmentation software is not optimized for the PROSE application.
Although preliminary, our results demonstrate the capability of PSI’s approach to generate accurate surface plots over relatively large areas of the eye, which is not currently possible with any other existing platform. Testing the technology on human volunteers is currently underway at Boston Foundation for Sight.
AO has achieved success in a range of applications in ophthalmology where microstructures need to be identified,
counted, and mapped. Multiple images are averaged to improve the SNR or analyzed for temporal dynamics. For small
patches, image registration by cross-correlation is straightforward. Larger images require more sophisticated registration
techniques. Strip-based registration has been used successfully for photoreceptor mosaic alignment in small patches;
however, if the deformations along long strips are not simple displacements, averaging will actually degrade the images.
We have applied non-rigid registration that significantly improves the quality of processed images for mapping cones
and rods, and microvasculature in dark-field imaging. Local grid deformations account for local image stretching and
compression due to a number of causes. Individual blood cells can be traced along capillaries in high-speed imaging
(130 fps) and flow dynamics can be analyzed.
The performance of clinical confocal SLO and OCT imagers is limited by ocular aberrations. Adaptive optics (AO)
addresses this problem, but most research systems are large, complex, and less well suited to the clinical environment.
PSI's recently developed compact retinal imager is designed for rapid, automated generation of cone photoreceptor
density maps. The device has a compact foot-print suitable for clinical deployment. The system previously presented at
Photonics West and ARVO 2013 has been upgraded to include numerous new features that support clinical research
applications. These upgrades significantly enhance the capabilities of the imager, providing the clinician with
simultaneously-acquired (registered) en face photoreceptor images and AO-OCT retinal cross-sections.
We describe here Mueller matrix microscopy, an imaging technique for investigating the anisotropic properties of the refractive index in biological samples. Tissue properties probed by polarization are the anisotropic real and imaginary parts of the refractive index that modify the polarization. Physical Sciences Inc. has developed a robust polarization microscope that is capable of performing complete Mueller matrix imaging in both transmission and reflection configuration. The system’s capabilities are illustrated on biological samples.
We recently developed several versions of a multimodal adaptive optics (AO) retinal imager, which includes highresolution
scanning laser ophthalmoscopy (SLO) and Fourier domain optical coherence tomography (FDOCT) imaging
channels as well as an auxiliary wide-field line scanning ophthalmoscope (LSO). Some versions have also been
equipped with a fluorescence channel and a retinal tracker. We describe the performance of three key features of the
multimodal AO system including: simultaneous SLO/OCT imaging, which allows SLO/OCT co-registration; a small
animal imaging port, which adjusts the beam diameter at the pupil from 7.5 to 2.5 mm for use with small animals
ubiquitous in biological research or for extended depth-of-focus imaging in humans; and slow scan Doppler flowmetry
imaging using the wide field auxiliary LSO imaging channel. The systems are currently deployed in several
ophthalmology clinics and research laboratories and several investigations have commenced on patients with a variety
of retinal diseases and animals in vision research.
Adaptive optics ophthalmic imaging systems that rely on a standalone wave-front sensor can be costly to build and
difficult for non-technical personnel to operate. As an alternative we present a simplified wavefront sensorless adaptive
optics laser scanning ophthalmoscope. This sensorless system is based on deterministic search algorithms that utilize the
image's spatial frequency as an optimization metric. We implement this algorithm on a NVIDIA video card to take
advantage of the graphics processing unit (GPU)'s parallel architecture to reduce algorithm computation times and
approach real-time correction.
We developed a multimodal adaptive optics (AO) retinal imager for diagnosis of retinal diseases, including glaucoma, diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinitis pigmentosa (RP). The development represents the first ever high performance AO system constructed that combines AO-corrected scanning laser ophthalmoscopy (SLO) and swept source Fourier domain optical coherence tomography (SSOCT) imaging modes in a
single compact clinical prototype platform. The SSOCT channel operates at a wavelength of 1 μm for increased penetration and visualization of the choriocapillaris and choroid, sites of major disease activity for DR and wet AMD.
The system is designed to operate on a broad clinical population with a dual deformable mirror (DM) configuration that
allows simultaneous low- and high-order aberration correction. The system also includes a wide field line scanning ophthalmoscope (LSO) for initial screening, target identification, and global orientation; an integrated retinal tracker (RT) to stabilize the SLO, OCT, and LSO imaging fields in the presence of rotational eye motion; and a high-resolution LCD-based fixation target for presentation to the subject of stimuli and other visual cues. The system was tested in a limited number of human subjects without retinal disease for performance optimization and validation. The system was able to resolve and quantify cone photoreceptors across the macula to within ~0.5 deg (~100-150 μm) of the fovea,
image and delineate ten retinal layers, and penetrate to resolve targets deep into the choroid. In addition to instrument
hardware development, analysis algorithms were developed for efficient information extraction from clinical imaging
sessions, with functionality including automated image registration, photoreceptor counting, strip and montage stitching, and segmentation. The system provides clinicians and researchers with high-resolution, high performance adaptive optics imaging to help guide therapies, develop new drugs, and improve patient outcomes.
An automated algorithm for differentiating breast tissue types based on optical coherence tomography (OCT) data is presented. Eight parameters are derived from the OCT reflectivity profiles and their means and covariance matrices are calculated for each tissue type from a training set (48 samples) selected based on histological examination. A quadratic discrimination score is then used to assess the samples from a validation set. The algorithm results for a set of 89 breast tissue samples were correlated with the histological findings, yielding specificity and sensitivity of 0.88. If further perfected to work in real time and yield even higher sensitivity and specificity, this algorithm would be a valuable tool for biopsy guidance and could significantly increase procedure reliability by reducing both the number of nondiagnostic aspirates and the number of false negatives.
To compare the optical properties of the human retina, 3-D volumetric images of the same eye are acquired with two nearly identical optical coherence tomography (OCT) systems at center wavelengths of 845 and 1060 nm using optical frequency domain imaging (OFDI). To characterize the contrast of individual tissue layers in the retina at these two wavelengths, the 3-D volumetric data sets are carefully spatially matched. The relative scattering intensities from different layers such as the nerve fiber, photoreceptor, pigment epithelium, and choroid are measured and a quantitative comparison is presented. OCT retinal imaging at 1060 nm is found to have a significantly better depth penetration but a reduced contrast between the retinal nerve fiber, the ganglion cell, and the inner plexiform layers compared to the OCT retinal imaging at 845 nm.
By increasing the speed and reducing the complexity of OCT systems, the parallel OCT system presented here will reduce the cost of high performance instruments while making them more widespread and easy to use. The instrument integrates line scanning with a swept source to achieve ultrahigh image speeds in a much simpler way. The swept source speed requirements are significantly relaxed. In a traditional OCT, a focused beam is scanned for image formation, while parallel OCT images simultaneously a full B-scan or a complete 3D cube. The system achieved ultrahigh scan speeds (98 kHz line rates and 160 fps), and was initially tested on various reflective and diffuse targets. The system demonstrates the potential for 3-D volumetric mapping of tissue at several volumes per second.
Subretinal neovascular membranes (SRNM) are a deleterious complication of laser eye injury and retinal diseases such as age-related macular degeneration (AMD), choroiditis, and myopic retinopathy. Photodynamic therapy (PDT) and anti-vascular endothelial growth factor (VEGF) drugs are approved treatment methods. PDT acts by selective dye accumulation, activation by laser light, and disruption and clotting of the new leaky vessels. However, PDT surgery is currently not image-guided, nor does it proceed in an efficient or automated manner. This may contribute to the high rate of re-treatment. We have developed a multimodal scanning laser ophthalmoscope (SLO) for automated diagnosis and image-guided treatment of SRNMs associated with AMD. The system combines line scanning laser ophthalmoscopy (LSLO), fluorescein angiography (FA), indocyanine green angiography (ICGA), PDT laser delivery, and retinal tracking in a compact, efficient platform. This paper describes the system hardware and software design, performance characterization, and automated patient imaging and treatment session procedures and algorithms. Also, we present initial imaging and tracking measurements on normal subjects and automated lesion demarcation and sizing analysis of previously acquired angiograms. Future pre-clinical testing includes line scanning angiography and PDT treatment of AMD subjects. The automated acquisition procedure, enhanced and expedited data post-processing, and innovative image visualization and interpretation tools provided by the multimodal retinal imager may eventually aid in the diagnosis, treatment, and prognosis of AMD and other retinal diseases.
We have developed a compact retinal imager that integrates adaptive optics (AO) into a line scanning laser ophthalmoscope (LSLO). The bench-top AO-LSLO instrument significantly reduces the size, complexity, and cost of research AOSLOs, for the purpose of moving adaptive optics imaging more rapidly into routine clinical use. The AO-LSLO produces high resolution retinal images with only one moving part and a significantly reduced instrument footprint and number of optical components. The AO-LSLO has a moderate field of view (5.5 deg), which allows montages of the macula or other targets to be obtained more quickly and efficiently. In a preliminary human subjects investigation, photoreceptors could be resolved and counted within ~0.5 mm of the fovea. Photoreceptor counts matched closely to previously reported histology. The capillaries surrounding the foveal avascular zone could be resolved, as well as cells flowing within them. Individual nerve fiber bundles could be resolved, especially near the optic nerve head, as well as other structures such as the lamina cribrosa. In addition to instrument design, fabrication, and testing, software algorithms were developed for automated image registration, cone counting, and montage stitching.
Accurate wavelength assignment of each spectral element for spectral-domain optical coherence tomography (SD-OCT) and optical frequency domain imaging (OFDI) is required for proper construction of biological tissue cross-sectional images. This becomes more critical for functional extensions of these techniques, especially in polarization-sensitive optical coherence tomography (PS-OCT), where incorrect wavelength assignment between the two orthogonal polarization channels leads to polarization artifacts. We present an autocalibration method for wavelength assignment that does not require separate calibration measurements and that can be applied directly on actual data. Removal of the birefringence artifact is demonstrated in a PS-OCT system with picometer accuracy in the relative wavelength assignment, resulting in a residual phase error of 0.25 deg/100 µm. We also demonstrate, for the first time, a quantitative birefringence map of an in vivo human retinal nerve fiber layer.
KEYWORDS: Signal to noise ratio, Retinal scanning, Retina, In vivo imaging, Optical coherence tomography, Motion measurement, Cameras, Standards development, Eye
We present pulsed illumination spectral-domain optical coherence tomography (SD-OCT) for in vivo human retinal imaging. We analyze the signal-to-noise (SNR) for continuous wave (CW) and pulsed illumination SD-OCT. The lateral beam scan motion is responsible for a SNR drop due to lateral scanning induced interference fringe washout. Pulsed illumination can reduce the SNR drop by shorter sample illumination time during the integration time of a camera. First, we demonstrate the SNR benefit of pulsed illumination over CW as function of lateral scan speed for a paper sample. For in-vivo human retinal imaging with pulsed illumination, the maximum permissible exposure (MPE) according to pulse repetition rate is presented based on ANSI standard. Finally, we show better SNR in retinal images of a normal subject with pulsed illumination SD-OCT over CW at high lateral scanning speed.
Spectral-Domain Optical Coherence Tomography (SDOCT) allows for in-vivo video-rate investigation of biomedical
tissue depth structure with the purpose of non-invasive optical diagnostics. In ophthalmic applications, it has been
suggested that Optical Coherence Tomography (OCT) can be used for diagnosis of glaucoma by measuring the thickness
of the Retinal Nerve Fiber Layer (RNLF). We present here an automated method for determining the RNFL thickness
map from a 3-D dataset. Boundary detection has been studied since the early days of computer vision and image
processing, and different approaches have been proposed. The procedure described here is based on edge detection using
a deformable spline (snake) algorithm. As the snake seeks to minimize its overall energy, its shape will converge on the
image contour, the boundaries of the nerve fiber layer. In general, the snake is not allowed to travel too much, and
therefore, proper initialization is required. The snake parameters, elasticity, rigidity, viscosity, and external force weight
are set to allow the snake to follow the boundary for a large number of retinal topographies. The RNFL thickness map is
combined with an integrated reflectance map of the retina and retinal cross-sectional images (OCT movie), to provide
the ophthalmologist with a familiar image for interpreting the OCT data. The video-rate capabilities of our SDOCT
system allow for mapping the true retinal topography since the motion artifacts are significantly reduced as compared to
slower time-domain systems.
Spectral-Domain Optical Coherence Tomography (SDOCT) allows for in-vivo video-rate investigation of biomedical tissue depth structure intended for non-invasive optical diagnostics. It has been suggested that OCT can be used for di-agnosis of glaucoma by measuring the thickness of the Retinal Nerve Fiber Layer (RNLF). We present an automated method for determining the RNFL thickness from a 3-D dataset based on edge detection using a deformable spline algo-rithm. The RNFL thickness map is combined with an integrated reflectance map and retinal cross-sectional images to provide the ophthalmologist with a familiar image for interpreting the OCT data. The video-rate capabilities of our SDOCT system allow for mapping the true retinal topography since motion artifacts are significantly reduced as com-pared to slower time-domain systems. Combined with Doppler Velocimetry, SDOCT also provides information on retinal blood flow dynamics. We analyzed the pulsatile nature of the bidirectional flow dynamics in an artery-vein pair for a healthy volunteer at different locations and for different blood vessel diameters. The Doppler phase shift is determined as the phase difference at the same point of adjacent depth profiles, and is integrated over the area delimited by two circles corresponding to the blood vessels location. Its temporal evolution clearly shows the blood flow pulsatile nature, the cardiac cycle, in both artery and vein. The artery is identified as having a stronger variation of the integrated phase shift. We observe that artery pulsation is always easily detectable, while vein pulsation seems to depend on the veins diameter.
Spectral-Domain Polarization-Sensitive Optical Coherence Tomography (SD-PS-OCT) is a technique developed to measure the thickness and birefringence of the nerve fiber layer in vivo as a tool for the early diagnosis of glaucoma. A clinical SD-PS-OCT system was developed and scans were made around the optic nerve head (ONH) using ten concentric circles of increasing diameter. One healthy volunteer was imaged. Retinal nerve fiber layer thickness and birefringence information was extracted from the data. Polarization-sensitive OCT images were acquired at video rate (29 frames per second (fps), 1000 A-lines / frame) and at 7 fps (1000 A-lines / frame). The last setting improved the signal to noise ratio by approximately 6 dB. Birefringence measurements on the healthy volunteer gave similar results as earlier reported values that were obtained with a time-domain setup. The measurement time was reduced from more than a minute to less than a second.
Microfluidic devices are becoming increasingly popular for many applications, enabling biological and chemical reactions to be performed with nano- and picoliter sample volumes. Accurate measurement and monitoring of fluid flow behavior in the small channels of microfluidic systems is important for evaluating the performance of existing devices, and in the modeling and design of new microfluidic networks. We present here the results of experiments using spectral-domain optical Doppler tomography (SD-ODT) to measure fluid flow in single-layer microfluidic devices. The principles behind flow imaging with SD-ODT are reviewed, a method for velocity calibration is described, and cross-sectional and en-face images of fluid velocity in microfluidic channels are presented.
Microfluidic devices are becoming increasingly popular for many applications, enabling biological and chemical reactions to be performed with nano- and picoliter sample volumes. Accurate measurement and monitoring of fluid flow behavior in the small channels of microfluidic systems is important for evaluating the performance of existing devices, and in the modeling and design of new microfluidic networks. We present here the results of experiments using spectral-domain optical Doppler tomography (SD-ODT) to measure fluid flow in single-layer microfluidic devices. The principles behind flow imaging with SD-ODT are reviewed, a method for velocity calibration is described, and cross-sectional and en-face images of fluid velocity in microfluidic channels are presented.
The polarimetric characteristics are experimentally investigated for dense scattering media such as compacted powders. The Mueller matrix is measured in backscattering configuration which is of interest for remote sensing as well as for characterizing rough scattering surfaces. Significant differences in various Mueller matrix elements are observed for different samples and are analyzed in terms of surface and volume scattering from an inhomogeneous medium.
In long-range applications, a laser beam propagating through atmosphere or particulate systems is depolarized due to multiple scattering. The transfer function of the state of polarization is given by the Mueller matrix associated to that system. We present here a systematic study of the polarization transfer function associated with multiple scattering systems. Real-time measurements of the Mueller matrix are presented for systems with various optical densities. We have specifically addressed the problem of noise reduction by overdetermining the Mueller Matrix. Experimental results of depolarization effects due to multiple scattering are shown for standard media that mimic atmospheric, plum or underwater propagation. This method aims at: (1) accounting for depolarization effects in long-range target identification and (2) remote monitoring and characterization of particulate systems.
The paper deals with development and realization of a heterodyne He-Ne laser Doppler system able to measure from distance the mechanical vibrations of a radar antenna. These vibrations, caused by wind and/or by proper system, could give false information concerning the velocity of a target. To measure the frequency and the amplitude of mechanical vibrations we used the optical heterodyne technique.
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