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We report first observations of the three-dimensional morphology of cone photoreceptors in the living human retina.
Images were acquired with a high-speed adaptive optics (AO) spectral domain optical coherence tomography (SD-OCT)
camera. The AO system consists of a Shack-Hartmann wavefront sensor and bimorph deformable mirror (AOptix) that
measure and correct the ocular and system aberrations at a closed-loop rate of 12 Hz. Unlike previous AO-OCT and AOSLO
instruments, the bimorph mirror was strategically positioned between the XY mechanical scanners and the subject's
eye so as to avoid beam distortion at the pupil plane, which is created when the mirror compensates for the refractive
error of the eye. This new configuration is evaluated empirically and with commercial ray tracing software. The SDOCT
system consists of a superluminescent diode and a 512 pixel line scan charge-coupled device (CCD) that acquires
75,000 A-scans/sec. This rate is more than two times faster than that previously reported. Retina motion artifiacts were
minimized by quickly acquiring small volume images of the retina with and without AO compensation. Camera
sensitivity was sufficient to detect reflections from all major retinal layers. The distribution of bright spots observed
within C-scans at the inner segment / outer segment (IS/OS) junction and at the posterior tips of the OS were found to be
highly correlated with one another and with the expected cone spacing. No correlation was found between the IS/OS
junction and either the plexiform layers or the layers immediately behind the OS posterior tips.
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The acquisition speed of current FD-OCT (Fourier Domain - Optical Coherence Tomography) instruments allows rapid
screening of three-dimensional (3D) volumes of human retinas in clinical settings. To take advantage of this ability
requires software used by physicians to be capable of displaying and accessing volumetric data as well as supporting
post processing in order to access important quantitative information such as thickness maps and segmented volumes.
We describe our clinical FD-OCT system used to acquire 3D data from the human retina over the macula and optic
nerve head. B-scans are registered to remove motion artifacts and post-processed with customized 3D visualization and
analysis software. Our analysis software includes standard 3D visualization techniques along with a machine learning
support vector machine (SVM) algorithm that allows a user to semi-automatically segment different retinal structures
and layers. Our program makes possible measurements of the retinal layer thickness as well as volumes of structures of
interest, despite the presence of noise and structural deformations associated with retinal pathology. Our software has
been tested successfully in clinical settings for its efficacy in assessing 3D retinal structures in healthy as well as
diseased cases. Our tool facilitates diagnosis and treatment monitoring of retinal diseases.
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We present three dimensional (3D) imaging of macular diseases and glaucoma with high speed, Fourier domain optical coherence tomography (FD-OCT). Our FD-OCT system allows video rate cross-sectional imaging with 98 dB sensitivity and 4.3 μm depth-resolution in tissue. This performance results in high contrast sectional images that enhance visualization of fine retinal layers including external limiting membrane and of deep structure such as the choroid and optic nerve. Volume rendering of 3D OCT data set taken for 3.5 seconds provides realistic 3D images of macular, optic disc and their pathologic changes. This manuscript will show the methods for three dimensional FD-OCT including a raster scanning protocol for volume rendering and cancellation of the motion artifact of eye balls, and the application of the high contrast three dimensional OCT imaging to macular diseases and glaucoma in clinical examination.
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Changes in cellular metabolism are considered first signs of fundus diseases, e.g. of age-related macular degeneration. Changes in the metabolism can potentially be detected by measuring the autofluorescence of the fundus. The fundus contains a wide variety of fluorophores in different binding and quenching states. The fluorescence signals cannot be clearly discriminated by commonly used steady state imaging techniques, even when these are combined with spectral resolution and excitation wavelength multiplexing. A considerable improvement is obtained by fluorescence lifetime imaging (FLIM). FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Due to its high sensitivity and high time resolution, its capability to resolve multi-exponential decay functions, and its easy combination with fast scanning we use multi-dimensional time-correlated single photon counting for fundus imaging. By analyzing the spectral properties of the expected fluorophores in the fundus, we show that improved discrimination of fluorophores is obtained by FLIM in combination with selected excitation wavelength and emission wavelength. As demonstrated in lifetime histograms of 40° fundus images, several fluorophores are excited at 446 nm, but predominantly lipofuscin at 468 nm excitation. Simultaneous detection of lifetime images in two emission ranges 500 nm to 560 nm and 560 nm to 700 nm improves further the discrimination of fluorophores.
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PURPOSE: Pharmacologic vitreolysis is a new approach to improve vitreo-retinal surgery. Ultimately, the development of drugs to liquefy and detach vitreous from retina should prevent disease by mitigating the contribution of vitreous to retinopathy and eliminate the need for surgery. However, the mechanism of action of pharmacologic vitreolysis remains unclear. The technique of Dynamic light scattering (DLS) was used to evaluate the effects of microplasmin by following the diffusion coefficients of spherical polystyrene nano-particles injected with microplasmin into the vitreous. METHODS: Diffusion coefficients in dissected (n=9) porcine eyes were measured in vitro. DLS was performed on all specimens at 37°C as often as every 10 minutes for up to 6 hours following injections of human recombinant microplasmin at doses ranging from 0.125 mg to 0.8 mg, with 20 nm diameter tracer nanospheres. RESULTS: DLS findings in untreated porcine vitreous were similar to the previously described findings in bovine and human vitreous, demonstrating a fast (early) component, resulting from the flexible hyaluronan molecules, and a slow (late) component, resulting form the stiff collagen molecules. Microplasmin increased porcine vitreous diffusion coefficients. A new approach was developed to use DLS measurements of vitreous diffusion coefficients to evaluate the effects of microplasmin in intact eyes. CONCLUSIONS: Pharmacologic vitreolysis with human recombinant microplasmin increases vitreous diffusion coefficients in vitro. The results of these studies indicate that this new approach using DLS to measure vitreous diffusion coefficients can be used to study the effects of pharmacologic vitreolysis using microplasmin and other agents in intact eyes and ultimately in vivo.
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Purpose: To identify, characterize, and discuss the current technological status of in vivo corneal diagnostic imaging and target high-priority future development needs. Methods: In vivo tandem scanning microscopy (non-coherent), scanning slit confocal microscopy (noncoherent), and laser scanning confocal microscopy (coherent) are examined. The current and future roles of multi-photon and higher order harmonic imaging are also discussed. Results and Conclusions: This keynote review demonstrates the current abilities and limitations of three currently used clinical imaging modalities to resolve the cellular and structural layers of the cornea temporally and spatially in three or four dimensions (x, y, z, t), with applications to the study of clinical-pathological processes such as inflammation; infection, wound healing, drug toxicity, organ development, differentiation and effects of genetic diseases. Each of these approaches has strengths and weaknesses. Thus, future technological development is essential to provide exciting new insights into understanding the structure and function of not only the cornea and the other ocular structures, but also other multicellular organs in health and disease. These imaging paradigms are among the most important advances in medical science in the past
three decades.
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We present results of ex vivo imaging of the mouse cornea following photorefractive keratectomy and in vivo imaging in the anterior segment of the rat eye using full-field optical coherence tomography. The instrument is based on the Linnik interferometer, illuminated by a white light source: a tungsten halogen lamp for ex vivo imaging and a fibered Xenon arc lamp for in vivo imaging. En face tomographic images are obtained in real-time without scanning by calculating the difference of two phase-opposed interferometric images recorded by a CCD or CMOS camera. Spatial resolution of ~1 μm in both axial and lateral directions is achieved thanks to the short coherence length of the illumination source and the use of relatively high numerical aperture microscope objectives. A detection sensitivity of up to 90 dB is reached by means of pixel binning and image averaging. Photorefractive keratectomy was performed on mice and the excised eyes were examined under immersion 21 days after surgery. Rats were anesthetized and their anterior segments imaged under immersion. The high resolution of our instrument gives cellular-level resolution in the cornea, allowing visualization of individual stromal keratocytes and collagen fibers, and cells in the endothelium. The basal and Descemet's membranes are well defined. Quantitative measurement of scattering in each layer is possible. Penetration to the level of the lens surface is achieved. Acquisition of stacks of en face images permits three-dimensional navigation through the cornea. Development of image treatment algorithms to allow three-dimensional reconstruction is discussed. The full-field optical coherence tomography technique could be useful in monitoring corneal scattering following refractive surgery.
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In the early stages of some retinal diseases, such as glaucoma, loss of retinal activity may be difficult to detect with today's clinical instruments. Many of today's instruments focus on detecting changes in anatomical structures, such as the nerve fiber layer. Our device, which is based on a modified fundus camera, seeks to detect changes in optical signals that reflect functional changes in the retina. The functional imager uses a patterned stimulus at wavelength of 535nm. An intrinsic functional signal is collected at a near infrared wavelength. Measured changes in reflectance in response to the visual stimulus are on the order of 0.1% to 1% of the total reflected intensity level, which makes the functional signal difficult to detect by standard methods because it is masked by other physiological signals and by imaging system noise. In this paper, we analyze the video sequences from a set of 60 experiments with different patterned stimuli from cats. Using a set of statistical techniques known as Independent Component Analysis (ICA), we estimate the signals present in the videos. Through controlled simulation experiments, we quantify the limits of signal strength in order to detect the physiological signal of interest. The results of the analysis show that, in principle, signal levels of 0.1% (-30dB) can be detected. The study found that in 86% of the animal experiments the patterned stimuli effects on the retina can be detected and extracted. The analysis of the different responses extracted from the videos can give an insight of the functional processes present during the stimulation of the retina.
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A functional extension of ultrahigh resolution OCT (UHR OCT) has been developed, that has the potential to establish this technique as an optical analogue to electrophysiology, by detecting depth resolved variations in optical backscattering caused by physiological tissue changes. This technique has been used to perform in vitro studies on excised, but physiologically intact, rabbit retinas and in vivo experiments on human retinas. UHR OCT has been synchronized with the white light stimulus to properly detected spatially resolved alterations in optical backscattering over time caused by lightinduced
intraretinal, physiological changes and has been correlated with simultaneous ERG recordings. Preliminary results demonstrate the potential of this novel extension of UHR OCT to detect time-dependent optical backscattering changes after application of a white light stimulus in specific retinal layers, especially in the inner and outer segments of the photoreceptor layer. Control experiments, including no light stimulus or application of drugs (in in vitro studies only) that inhibit the physiological responses of certain type of retinal cells confirm the physiological origin of the
detected backscattering changes. Detection of cell activity and cell physiology by UHR OCT would enable a better understanding of basic physiological phenomena and may also contribute to better understanding of retinal pathogenesis.
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Non-invasive in vivo functional optical imaging is emonstrated using high-speed, ultrahigh resolution optical coherence tomography (UHR-OCT). A high-speed, UHR-OCT system using spectral/Fourier domain detection was developed for functional imaging experiments in the rodent retina. Using a spectrally multiplexed superluminescent diode light source, imaging was performed with 2.8 μm resolution at a rate of 24,000 axial scans per second. OCT measurement protocols were designed to minimize noise sources that cause undesired fluctuations in the measured OCT signal. A white light stimulus was applied to the retina and the average reflectivity from each intraretinal layer was monitored over time using OCT. A white light stimulus induces a response consisting of an increase in the reflectance of the photoreceptor outer segments. To our knowledge, this is the first in vivo demonstration of functional imaging using OCT in the retina. Further systematic investigation will be required to fully characterize the observed optical changes. Eventually, this may prove to be an objective method for measuring photoreceptor function in the human retina.
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A polarization sensitive optical coherence tomography (PS-OCT) instrument was used to investigate the retinal pigment epithelium (RPE). The instrument uses the polarization properties of light to record backscattered intensity, retardation and fast axis orientation simultaneously and needs only one measurement per sample location to retrieve these parameters. The polarization state of light backscattered from within the RPE was found to be random. This can be observed in PS-OCT images by random retardation and axis orientation values within the RPE layer. In diseased eyes where the normal retinal structure is corrupted (e.g. RPE atrophy, RPE detachment) the localization of the RPE within OCT images which do not provide polarization information (standard OCT) is rather difficult. Since the RPE is the only structure within the retina to cause this polarization scrambling, PS-OCT can be used for contrast enhancement and enables the exact localization of the RPE in these pathologies. Therefore it is possible to determine if the RPE is still preserved in regions of interest. Furthermore, in patients with RPE atrophy an enhanced penetration depth into the choroid and even into the sclera was observed. Because of birefringence introduced by the sclera the border between choroid and sclera could easily be determined.
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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.
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The effectiveness of the corneal ablation process in refractive surgery is mostly evaluated by indirect measures of vision or optical quality such as post-operative refraction or wavefront aberrometry. Yet, the effective amount of corneal tissue removed in the treatment can only be determined by correctly overlapping a pre- and a post-operative topography measurement. However such an overlap is not trivial due to the discrepancy in the centration axes used in the measurement and the treatment, as well as due to the shift of ocular axes through the treatment or tilt between the two surfaces. We therefore present two methods for overlapping pre- and post-operative topographies for the purpose of extracting an effective corneal ablation profile. Method one uses a 3-dimensional profile matching algorithm and cross-correlation analysis on surface rings outside the optical zone of the topographies. Method two employs a surface normal matching routine to align the two surfaces along their common ablation axis. The profile matching method implies the problem that it requires measurement data outside of the optical zone which was found to be uncertain with placido-disk-based topographers. Method number two is more simple and implies the advantage of using measurement data within the optical zone. For regular profiles the extracted ablation profiles showed a very good match with the planned ones. Surprisingly, even for highly irregular profiles of topography-guided laser treatments the method delivered reasonable overlaps when being compared to the planned profiles. Analysis of the effective tissue removal yields valuable information on the quality of the ablation process.
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The purpose of this work is to demonstrate the combination of reflective confocal microscopy and multiphoton
microscopy and its application in imaging cornea. The difficulty of optically imaging the highly translucent cornea has
prevented the development of an effective non-invasive system for the clinical monitoring of the physiological or
pathological states of corneas. In this work, we combine reflective confocal microscopy with multiphoton microscopy
to demonstrate the potential of our methodology in the minimally invasive imaging of the cornea. The visible reflection
signals from cornea can provide structural information of interfaces of different refractive indices while the multiphoton
signals generated from the use of near infrared excitation allows deep tissue penetration and reduced photo-damage. In
multiphoton imaging, the second harmonic generation (SHG) signal is used to detect collagen in the stroma of the cornea,
and the reflective confocal imaging allows detection of the cellular components located in the epithelium. The
combination of reflective and multiphoton imaging can be used to reveal complementary structural information of the
corneal architecture.. The system is first tested on porcine eye cornea. Assessment of the result on the porcine eye will be
used to evaluate the potential of the system as a technique for in vivo clinical applications.
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The purpose of this study is to assess the possible application of multiphoton fluorescence and second harmonic
generation (SHG) microscopy for imaging the structural features of keratoconus cornea and to evaluate its potential as
being a clinical in vivo monitoring technique. Using the near-infrared excitation source from a titanium-sapphire laser
pumped by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton
autofluorescence and SHG signals from the cornea specimens with keratoconus. A home-modified commercial
microscope system with specified optical components is used for optimal signal detection. Keratoconus cornea button
from patient with typical clinical presentation of keratoconus was obtained at the time of penetrating keratoplasty. The
specimen was also sent for the histological examination as comparison. In all samples of keratoconus, destruction of
lamellar structure with altered collagen fiber orientation was observed within whole layer of the diseased stromal area. In
addition, the orientation of the altered collagen fibers within the cone area shows a trend directing toward the apex of the
cone, which might implicate the biomechanical response of the keratoconus stroma to the intraocular pressure. Moreover,
increased autofluorescent cells were also found in the cone area, with increased density as one approaches the apical area.
In conclusion, multiphoton autofluorescence and SHG microscopy non-invasively demonstrated the morphological
features of keratoconus cornea, especially the structural alternations of the stromal lamellae. We believe that in the future
the multiphoton microscopy can be applied in vivo as an effective, non-invasive diagnostic and monitoring technique for keratoconus.
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As a part of an ongoing project on corneal endothelium morphometry by diffraction, a model for corneal endothelium simulation has been developed. The model has been developed in the mathematical programming language Matlab. Images of corneal endothelium were simulated and the diffraction pattern of the image was calculated. The diffraction pattern was calculated for a series of endothelial images while varying important variables in the simulated image. This rendered the theoretical relationships between values of variables in the diffraction pattern and values of morphometric variables in the image. At this stage, the analysis focused on the expression of endothelial mean cell size and coefficient of variation in the diffraction pattern, respectively. As expected from diffraction theory, it was found that there is a direct linear relationship between mean cell size and distance between periodic variations in the diffraction pattern. We further found that the ratio between the intensity in the central maximum and the intensity in the first harmonic of the diffraction pattern was functionally depending on the variation in cell size. The current findings demonstrate that it is possible to theoretically determine average cell size and coefficient of variation of cell size in the diffraction pattern.
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The viscoelastic properties of the cornea are important determinants of the corneal response to surgery and disease. The purpose of this work is to develop an OCT-based technique for non-contact, high-resolution pan-corneal strain mapping using clinically-achievable pressure changes as a stressor. Porcine corneas were excised and mounted on an artificial anterior chamber that facilitated maintenance of a simulated intraocular pressure (IOP). Pressure was controlled and monitored continuously by saline infusion with an in-line transducer and digital monitor. Mounted specimens were positioned under a laboratory-based high-speed OCT system and imaged in three dimensions at various IOP levels. Matlab and C++ routines were written to perform 2-D bitmap cross-correlation analyses on corresponding images at different pressure levels. Resulting correlations produced a likelihood estimate of the 2-D vector displacement of corneal optical features. Strain maps from cross-correlation analyses revealed local areas of highly consistent displacements interspersed with inter-regional variability. Displacements occurred predominantly along axial vectors. Our analysis produces results consistent with expected and observed displacement of the cornea with varying IOP. Cross-correlation analysis of optical feature flow in the corneal stroma can provide high-resolution strain maps capable of distinguishing spatial heterogeneity in the corneal response to pressure change. A non-destructive, non-contact technique for corneal strain mapping offers numerous potential advantages over tensile testing of excised tissue strips for inferring viscoelastic behavior, and the membrane inflation model employed here could potentially be extended to clinical biomechanical characterizations.
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Electronic retinal prostheses represent a potentially effective approach for restoring some degree of
sight in blind patients with retinal degeneration. However, levels of safe electrical stimulation and the
underlying mechanisms of cellular damage are largely unknown. We measured the threshold of cellular
damage as a function of pulse duration, electrode size, and number of pulses to determine the safe range of
stimulation. Measurements were performed in-vitro on embryonic chicken retina with saline-filled glass
pipettes for stimulation electrodes. Cellular damage was detected using Propidium Iodide fluorescent
staining. Electrode size varied from 115μm to 1mm, pulse duration from 6μs to 6ms, and number of pulses
from 1 to 7,500. The threshold current density was independent of electrode sizes exceeding 400μm. With
smaller electrodes the current density was scaling reciprocal to the square of the pipette diameter, i.e. acting
as a point source so that the damage threshold was determined by the total current in this regime. The
damage threshold current measured with large electrodes (1mm) scaled with pulse duration as t-0.5, which is
characteristic of electroporation. For repeated electrical pulsed exposure on the retina the threshold current
density varied between 0.059 A/cm2 at 6ms to 1.3 A/cm2 at 6μs. The dynamic range of safe stimulation,
i.e. the ratio of damage threshold to stimulation threshold was found to be duration-dependent, and varied
from 10 to 100 at pulse durations varying between 10μs to 10ms. Maximal dynamic range of 100 was
observed near 1ms pulse durations.
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We present a multi-chip electric stimulator for a retinal prosthesis. The stimulator consists of small silicon devices (unit chips) molded in a thin film. It has an advantage over the conventional devices in physical flexibility and extendibility. The smart unit chip (600 μm square, in this work) is an integrated circuit (IC) that includes digital serial interface circuits, analog switch circuits and on-chip stimulus electrodes. In contrast to conventional stimulators, the present stimulator can be driven with only four wires. The multi-chip configuration enables to make the stimulator flexible and durable to bending stress. The device can be bended to place the stimulation electrodes in good contact with retinal tissue. In this paper, we present the design of the stimulator device with 0.35-μm complementary metal-oxide semiconductor (CMOS) technology. We also report a thin, flexible packaging technique for the stimulator and preliminary experimental results of a sputtered iridium oxide (IrOx) film that can be used for chronic stimulation.
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One of the major complications of cataract surgery is posterior capsule opacification caused by proliferation and migration of residual lens epithelial cells into the visual axis. In this study we present a novel approach to treat posterior capsule opacification in a non-invasive manner. A polymer-drug conjugate has been developed which is suitable for manufacturing functional intraocular lenses equipped with a drug delivery system. The therapeutic molecules, 5-fluorouracil, were attached through a photolabile linkage to the acrylic polymer backbone of the intraocular lens material. The controlled release of 5-fluorouracil is accomplished by two-photon induced cleavage of the linkage which is stable in ordinary conditions. The properties of the therapeutic system are characterized and the function is demonstrated in in vitro tests. The utilization of two-photon-absorption processes in drug delivery may provide a powerful tool to prevent posterior capsule opacification.
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Purpose: To assess the efficacy of a novel orbital tissue expander (OTE) in treating congenital anophthalmic and microphthalmic infants. Methods: The OTE implant is an inflatable (0.5 to >6cc) silicone rubber globe sliding on a titanium T-shaped bone plate secured to the temporal bone with 1mm titanium screws. In vitro testing was performed to assess injection volume versus diameter measurements to determine consistency between devices, flex fatigue for durability of the implants when compressed, weight change in isotonic saline at 37°C to mimic human body temperature, seal durability by puncturing the globe numerous times while inflating, capacity before rupture to determine the maximum amount of saline it is able to contain, and effective sterilization. Ex-vivo testing was performed for adjustments prior to in vivo study. An OTE was then implanted in five 2-week old kittens (OS only) and inflated in 0.5cc increments. Three control animals received enucleation alone. All 8 animals were followed for 18 weeks and underwent euthanasia for morphological and histopathological analysis. Results: In vitro testing confirmed a <5% diameter variance between different OTEs inflated in 0.5cc increments up to 5.0cc, <5% weight change in isotonic saline at 37°C over 7 weeks, <3% weight change over 14 months in the fatigue tester, and no quantifiable leakage (<1mg) after 65 consecutive 30ga needle punctures. The OTEs were successfully sterilized by autoclave and easily secured in the orbit of postmortem kittens. The in vivo study demonstrated biocompatibility of the implant which stimulates orbital bone growth resulting in almost no difference between the implanted socket and the control eye of the cat. There were no adverse effects in the normal maturation, weight gain, and food intake of the cats. Light microscopy showed no signs of foreign body reaction. Pictures of the implants were obtained by using a shadow-photogrammetry system to compare the explanted OTE with the OD control eye. Conclusion: In vitro and in vivo studies show the implant's potential to safely treat anophthalmic and microphthalmic infants.
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Purpose: To design and test the Miami-InnFocus Drainage Implant (MiDi) as a glaucoma shunt that is biocompatible,
flexible, and significantly smaller than existing commercial implants in order to prevent postoperative hypotony, inflammation, scarring, erosion, and extrusion.
Methods: A new biomaterial composed of styrene-b-isobutylene-b-styrene (SIBS) was used in a novel design for a glaucoma drainage implant. The implant consists of a tube 11mm long with an inner diameter of 70, 100, and 150 μm
and outer diameter of 250 μm with a 1mm2 tab located 4.5mm from the proximal tip to prevent migration. The device
was implanted in 15 New Zealand White rabbits for biocompatibility and efficacy testing. A similarly designed implant
made of polydimethylsiloxane was implanted in 6 other animals as a pseudo-control. Typical GDI implantation
technique was modified for this device. The proximal end of the new SIBS implant was inserted 2mm into the anterior
chamber and the distal end placed in a subconjunctival space created by the surgeon. Biocompatibility of the device was
studied by slit-lamp follow-up and intraocular pressure (IOP) measurements recorded periodically. Results:
Biocompatibility of the MiDi was excellent. A low and diffuse bleb was observed with these devices. All SIBS tubes
were patent 9 months after insertion. Immunostaining demonstrated non-continuous deposition of collagen with virtually
no encapsulation. No macrophages or myofibroblast were visible around the SIBS polymer which was found more
bioinert than the control PDMS.
Conclusion: This newly designed glaucoma implant is clinically biocompatible in the rabbit model and maintained
100% patency at 9 months.
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We are developing a non- or minimally-invasive method for detecting and measuring specific drugs and biomolecules in vivo using photoacoustic spectroscopy (PAS). This pilot study investigated the feasibility of detecting the concentration of certain drugs in the vitreous or aqueous of the eye. As a prototype for using PAS for molecular detection in vivo, the technique was applied to the detection in a surrogate eye, of drugs with known optical spectrum such as Trypan Blue, Rose Bengal, and Amphotericin B (AB), at concentrations as low as 1 μg/ml. Chopped CW, or short pulse, Q-switch lasers, were used as pumping sources to generate ultrasonic photoacoustic signals in an ocular phantom containing the drug solutions. In addition to an ultrasonic hydrophone, the photothermal deflection technique (PhDT), a non-contact optical method with high sensitivity and fast response, were used to record the photoacoustic signals. The data from both detectors were compared over a range of drug concentrations. The photoacoustic signal generated from the retina was used as a reference, to measure the attenuation of light through drug solutions of different concentrations in the ocular phantom. The results indicated that photoacoustic spectroscopy is feasible in ocular phantoms incorporating ex vivo ocular tissue. The signals recorded using PAS were to be found to be linearly dependent on drug concentration, as predicted by theory. The photoacoustic method was found to be sensitive to drug concentrations as low as 1 μg/ml, a clinically relevant concentration for many drugs. Future work will be directed at adapting this method for in vivo measurement, and enhancing its sensitivity by using a tunable laser as the pump source.
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The advancement in adaptive optics in recent years has increased the interest in the dynamic aberrations of the eye, including those introduced by the first optical surface provided by the tear film. A curvature sensing system to measure the dynamic topography of the tear film is described. This optical system was used to measure the
aberrations of the tear film on 14 eyes. The evolution of this surface is monitored through videos of the tear film topography. The effect on optical quality is studied from the time-evolution of the RMS wavefront error showing non-negligible aberration variations attributed to the tear film layer; the effect of tear film break-up on the ocular optical quality is also discussed. Furthermore, the aberration maps are decomposed into their constituent Zernike
components showing stronger contributions from 4th order terms, and also from those components with vertical symmetry which can be attributed to the effect of the eye lids on the tear film. Finally, the power spectra of the RMS wavefront error evolution show that the strongest contributions of the tear film aberrations are to be found at low frequencies, typically below 2Hz.
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Numerous types of wavefront correctors have been employed in adaptive optics (AO) systems for correcting the wave aberrations of the eye. While each has been shown to reduce the degrading impact of the ocular aberrations, none have shown sufficient correction to yield diffraction-limited imaging for large pupils (≥6 mm), where the aberrations are most severe and the benefit of AO is largest. As the wavefront corrector appears to be the limiting AO component, it raises a fundamental concern as to what characteristics of this device, in particular actuator stroke and number, are required to achieve diffraction-limited imaging, and to optimally match corrector performance and cost to that required of a particular imaging task in the eye.
In this paper, we model the performance of discrete actuator deformable mirrors, piston-only segmented mirrors, and piston/tip/tilt segmented mirrors in conjunction with wavefront aberrations measured on human eyes in two large population datasets (University of Rochester and Indiana University). The actuator stroke and number required to achieve diffraction-limited imaging for a 7.5 mm pupil were found to be highly dependent on the level of 2nd order aberrations and the population considered. Specifically, the required stroke for encompassing 95% of the population ranged from 12-53 (Rochester) and 7-11 (Indiana) microns. The wide range resulted from whether 2nd order aberrations were corrected or set to zero prior to correction. To achieve a Strehl > 0.8, the actuator number across the pupil diameter ranged from >14 (Rochester) and 11-14 (Indiana) for discrete actuator deformable mirrors, >95 (Rochester) and 50-90 (Indiana) for piston-only segmented mirrors, and finally 12-19 (Rochester) and 9-10 (Indiana) for piston/tip/tilt segmented mirrors.
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Three-dimensional ultrahigh resolution optical coherence tomography (UHR OCT) and adaptive optics (AO) are combined using a liquid crystal programmable phase modulator (PPM) as a correcting device for the first time. AO is required for correcting ocular aberrations in moderate and large pupils in order to achieve high resolution retinal images. The capabilities of the PPM are studied using polychromatic light. Volumetric UHR OCT images of the living retina with AO, obtained with up 25000 A scans/s and high resolution (~5x5x3 μm; transverse (x) x transverse (y) x axial) are recorded, enabling visualization of interesting intraretinal morphological structures. Cellular retinal features, which might correspond to groups of terminal bars of photoreceptors at the level of the external limiting membrane, are resolved. Benefits and limitations of the presented technique are finally discussed.
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A MEMS deformable mirror (DM)-based new generation adaptive optics scanning laser ophthalmoscope (AOSLO) has been developed for in-vivo microscopic imaging of the living human retina. With the miniaturized optical aperture of a μDMS-MultiTM MEMS DM made by Boston Micromachines Corporation (Watertown, MA), we were able to confine a compact and robust optical system to a mobile 30"×30" breadboard while keeping the system aberrations diffraction-limited over an imaging field of view up to 3×3 degrees. A customized Shack-Hartmann wavefront sensor was devised to facilitate the MEMS DM based adaptive optics (AO) system. The ocular aberration is compensated over a 6mm pupil based upon a modal wavefront correction strategy. The AO correction is done for both ingoing and outgoing paths of the scanning laser ophthalmoscope. After AO correction, the root mean square wave aberration is reduced to less than 0.1μm for most eyes. The lateral resolution is effectively enhanced and the images reveal clear cone mosaic near the foveal center. The significant increase of the throughput at the confocal pinhole allows for a confocal pinhole whose diameter is less than the Airy disc of the collection lens, thereby fully exploiting the axial resolution capabilities of the system. The MEMS DM as well as its successful application represents the most significant technological breakthrough of this new generation AOSLO.
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Active image stabilization for an adaptive optics scanning laser ophthalmoscope (AOSLO) was developed and tested in
human subjects. The tracking device, a high speed, closed-loop optical servo which uses retinal features as tracking target, is separate from AOSLO optical path. The tracking system and AOSLO beams are combined via a dichroic beam
splitter in front of the eye. The primary tracking system galvanometer mirrors follow the motion of the eye. The AOSLO raster is stabilized by a secondary set of galvanometer mirrors in the AOSLO optical train which are "slaved"
to the primary mirrors with fixed scaling factors to match the angular gains of the optical systems. The AO system (at
830 nm) uses a MEMS-based deformable mirror (Boston Micromachines Inc.) for wave-front correction. The third
generation retinal tracking system achieves a bandwidth of greater than 1 kHz allowing acquisition of stabilized AO
images with an accuracy of <10 μm. However, such high tracking bandwidth, required for tracking saccades, results in
finite tracking position noise which is evident in AOSLO images. By means of filtering algorithms, the AOSLO raster is
made to follow the eye accurately with reduced tracking noise artifacts. The system design includes simultaneous presentation of non-AO, wide-field (~40 deg) live reference image captured with a line scanning laser ophthalmoscope
(LSLO) typically operating from 900 to 940nm. High-magnification (1-2 deg) AOSLO retinal scans easily positioned
on the retina in a drag-and-drop manner. Normal adult human volunteers were tested to optimize the tracking
instrumentation and to characterize AOSLO imaging performance. Automatic blink detection and tracking re-lock,
enabling reacquisition without operator intervention, were also tested. The tracking-enhanced AOSLO may become a
useful tool for eye research and for early detection and treatment of retinal diseases.
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Precise targeting of retinal structures including retinal pigment epithelial cells, feeder vessels, ganglion cells, photoreceptors, and other cells important for light transduction may enable earlier disease intervention with laser therapies and advanced methods for vision studies. A novel imaging system based upon scanning laser ophthalmoscopy (SLO) with adaptive optics (AO) and active image stabilization was designed, developed, and tested in humans and animals. An additional port allows delivery of aberration-corrected therapeutic/stimulus laser sources. The system design includes simultaneous presentation of non-AO, wide-field (~40 deg) and AO, high-magnification (1-2 deg) retinal scans easily positioned anywhere on the retina in a drag-and-drop manner. The AO optical design achieves an error of <0.45 waves (at 800 nm) over ±6 deg on the retina. A MEMS-based deformable mirror (Boston Micromachines Inc.) is used for wave-front correction. The third generation retinal tracking system achieves a bandwidth of greater than 1 kHz allowing acquisition of stabilized AO images with an accuracy of ~10 μm. Normal adult human volunteers and animals with previously-placed lesions (cynomolgus monkeys) were tested to optimize the tracking instrumentation and to characterize AO imaging performance. Ultrafast laser pulses were delivered to monkeys to characterize the ability to precisely place lesions and stimulus beams. Other advanced features such as real-time image averaging, automatic highresolution mosaic generation, and automatic blink detection and tracking re-lock were also tested. The system has the potential to become an important tool to clinicians and researchers for early detection and treatment of retinal diseases.
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A new significantly redesigned version of clinically applicable adaptive optics multispectral fundus imager is presented. Along with greatly improve adaptive system loop rate, the device performs reliably and is convenient for use in clinical practice. This new imager has allowed us to use new approaches for retina image analysis and obtain original results on the distribution of aberrations in the human eye.
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In this paper we consider anisoplanatism effect as a fundamental limitation on the size of high resolution area
(isoplanatic patch) of retinal images obtained using fundus cameras equipped with adaptive optics. Isoplanatic patch
size was estimated using experimental results for on-axis and off-axis eye aberrations measured by Shack-Hartmann
technique. Isoplanatic patch size varied among examined subjects in the range from 1.5o to 2.5o which is in good
agreement with results obtained using ray-tracing technique1. We estimated isoplanatic patch size for Gullstrand eye
model and found it to be close to the values obtained from experimental results for subjects with good vision. We also
discuss the possibilities of Gullstrand eye model modifications for modeling anisoplanatism effect for each particular
subject. We also estimated the efficiency of multibeacon correction method and found out that this method allows us to
almost twice increase the area with high resolution.
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Near infrared characterization of optical properties of various tissue components of healthy human and bovine eyes has been performed. The indices of refraction (n) of these ocular tissues were determined using a Michelson interferometer. The total diffuse reflection (Rd) and total transmission (Tt) measurements have been taken for individual ocular tissue by using double-integrating spheres and infrared laser diodes. The Inverse Adding Doubling computational method based on the diffusion approximation and radiative transport theory is applied to the measured values of n, Rd, and Tt to calculate the optical absorption and scattering coefficients of the human and bovine ocular tissues. The scattering anisotropy value was determined by iteratively running the inverse adding doubling method program and a Monte Carlo simulation of light-tissue interaction until the minimum difference in experimental and computed values for Rd and Tt were realized. A comparison between the optical characterization of human and bovine ocular samples is also
made.
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The most probable reason for presbyopia is an age related loss of elasticity of the lens. It progresses typically during the whole life and at the age of about 45 it leads to a considerable loss of the ability to accommodate within the next decade. However, both, the ciliary muscle and the lens capsule stay active and elastic, respectively. With respect to this, one concept is to regain the deformability of the lens without changing the capsule or zonular apparatus. Since the investigations of Ripken et al. proofed that the flexibility of the presbyopic lens tissue can be increased through the creation of fs-laser induced microcuts inside the lens, this is one possible approach to treat presbyopia. On this account a finite-element-method model with ANSYS of the human lens during accommodation will be presented. The analysis premises all lens materials to be linear elastic and allow large displacements. A first analysis of this method for the treatment of presbyopia is accomplished. Therefore the mechanical analysis of untreated and treated lens are compared. In addition ex-vivo elasticity measurements of untreated and treated lenses will be presented. As a result an improvement of the flexibility of the lens tissue is found and as its consequence a change of the lens radii of curvature is established. After suitable processing of the output data the change in optical power between untreated and treated lenses are calculated. The finite element simulation shows similar behaviour compared to the treated porcine lenses.
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Tumor thermo treatment such as photodynamic therapy (PDT) or transpupillary thermotherapy (TTT) deal with long term and large laser spot exposures. The induced temperature increase is not exactly known [1]. Under these conditions convective heat transfers due to the blood flow in the choroid and the choriocapillaris must be considered in addition to the usually calculated heat conduction. From an existing analytical model defining a unique convective term for the whole fundus irradiated with Gaussian irradiance distribution lasers [2], we developed a numerical one allowing a precise modelling of convection and calculating heating evolution and temperature profiles of the fundus of the eye. The aim of this study is to present the modelling and several comparisons between experimental results [3] and numerical ones concerning the convective heat transfers inside the fundus of the eye.
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We have developed a virtual reality (VR) simulator for phacoemulsification (phaco) surgery. The current work aimed at
evaluating the precision in the estimation of response variables identified for measurement of the performance of VR
phaco surgery. We identified 31 response variables measuring; the overall procedure, the foot pedal technique, the
phacoemulsification technique, erroneous manipulation, and damage to ocular structures. Totally, 8 medical or
optometry students with a good knowledge of ocular anatomy and physiology but naive to cataract surgery performed
three sessions each of VR Phaco surgery. For measurement, the surgical procedure was divided into a sculpting phase
and an evacuation phase. The 31 response variables were measured for each phase in all three sessions. The variance
components for individuals and iterations of sessions within individuals were estimated with an analysis of variance
assuming a hierarchal model. The consequences of estimated variabilities for sample size requirements were
determined. It was found that generally there was more variability for iterated sessions within individuals for
measurements of the sculpting phase than for measurements of the evacuation phase. This resulted in larger required
sample sizes for detection of difference between independent groups or change within group, for the sculpting phase as
compared to for the evacuation phase. It is concluded that several of the identified response variables can be measured
with sufficient precision for evaluation of VR phaco surgery.
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We present a preliminary study of a new method, based on the laser welding of suitably prepared patches of capsular
tissue for the closure of capsulorhexes in the lens capsule. This technique is proposed for the repair capsular breaks or
tears caused by accidental traumas or ones produced intraoperatively during standard IOL implantation. Experiments
were carried out ex vivo on freshly enucleated porcine eyes. Patches of anterior capsular tissue, collected from donor
eyes, were stained with a solution of Indocyanine Green (ICG) in sterile water. Closure tests on a capsulorhexis were
performed by welding a stained patch onto the recipient capsule, using diode laser radiation at 810 nm, which greatly
absorbed by the ICG-stained tissue. Laser radiation was delivered by means of a 200-micron-core-fiber, the tip of which
was gently pressed onto the patch surface (contact welding technique) so as to produce effective tissue welding in
underwater conditions. Laser-welded capsular tissue was found to have good resistance to mechanical load, comparable
in fact to that of healthy tissue.
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Before an intraocular lens (IOL) is implanted during cataract surgery, biometric data of the patient's eye have to be determined to calculate the thickness and shape of the IOL. In particular the postoperative anterior chamber depth is an important parameter to predict the correct shape of the IOL. This value, however, cannot be measured without significant uncertainities. We present a solution to this problem, describe novel polymers suitable for IOLs which refractive indices can be changed non-invasively in a photo-induced process. The focal length can be modified by about 2 D, which is sufficient to achive ideal acuteness of vision for almost all patients with implanted IOLs. The change in refractive index is accomplished by linking or cleaving bonds between a sufficiently large number of side groups of the polymer main chain in a photoinduced cyloaddition or cycloreversion, respectively. The photochemical reaction can also be triggered by a two-photon process (TPA) using a pulsed laser system, i.e. the energy required for bond breaking is provided by two photons in the visible range. Light in the UV as well as the visible range of the spectrum cannot induce undesired changes of the refractive index owing to the strong UV-absorption of the cornea and photon densities much too low for TPA, respectively. Due to the excellent spatial resolution that can be achieved with two-photon processes not only modification of the refractive index of the entire lens but also selectively in well defined areas is possible enabling the correction for aberrations such as astigmatism.
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A major obstacle in applying gene therapy to clinical practice is the lack of efficient and safe gene delivery
techniques. Viral delivery has encountered a number of serious problems including immunological reactions and
malignancy. Non-viral delivery methods (liposomes, sonoporation and electroporation) have either low efficiency in-vivo
or produce severe collateral damage to ocular tissues.
We discovered that tensile stress greatly increases the susceptibility of cellular membranes to electroporation.
For synchronous application of electric field and mechanical stress, both are generated by the electric discharge itself. A pressure wave is produced by rapid vaporization of the medium. To prevent termination of electric current by the vapor cavity it is ionized thus restoring its electric conductivity. For in-vivo experiments with rabbits a plasmid DNA was injected into the subretinal space, and RPE was treated trans-sclerally with an array of microelectodes placed outside the eye. Application of 250-300V and 100-200 μs biphasic pulses via a microelectrode array resulted in efficient
transfection of RPE without visible damage to the retina.
Gene expression was quantified and monitored using bioluminescence (luciferase) and fluorescence (GFP) imaging. Transfection efficiency of RPE with this new technique exceeded that of standard electroporation by a factor
10,000. Safe and effective non-viral DNA delivery to the mammalian retina may help to materialize the enormous
potential of the ocular gene therapy. Future experiments will focus on continued characterization of the safety and
efficacy of this method and evaluation of long-term transgene expression in the presence of phiC31 integrase.
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Transpupillary thermo therapy (TTT) is a slow (60 seconds) photothermal treatment of the fundus with a near-infrared (780-810nm) laser irradiating a large spot (0.5- 1. mm) on the retina. Due to high variability in ocular tissue properties and the lack of immediately observable outcome of the therapy, a real-time dosimetry is highly desirable. We found that fundus spectroscopy and spectrally-resolved imaging allow for non-invasive real-time monitoring and dosimetry of TTT. A 795nm laser was applied in rabbit eyes for 60 seconds using a 0.86mm retinal spot diameter. The fundus was illuminated with a broadband polarized light, and its reflectance spectra were measured in parallel and cross-polarizations. The fundus was also imaged in selected spectral domains. At irradiances that do not create ophthalmoscopically visible lesions the fundus reflectance increases at the wavelengths corresponding to absorption of the oxygenated blood indicating the reduced concentration of blood in the choroid. Vasoconstrictive response of the choroidal and retinal vasculature during TTT was also directly observed using spectrally-resolved imaging. At irradiances that produce ophthalmoscopically visible lesions a rapid reduction of the fundus reflectance was observed within the first 5-10 seconds of the exposure even when the visible lesions developed only by the end of the 60 second exposure. No visible lesions were produced where the laser was terminated after detection of the reduced scattering but prior to appearance of the enhanced scattering.
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In almost all retinal laser treatments the therapeutic effect is initiated by a transient temperature increase. Due to differences in tissue properties and physiology like pigmentation and vascular blood flow an individually different temperature increase might occur with crucial effects on the therapeutic benefit of the treatment. In order to determine the individual retinal temperature increase during cw-laser irradiation in real-time we developed a non-invasive method based on optoacoustics. Simultaneously to the cw-laser irradiation (λ = 810 nm, P < 3 W, t = 60 s) pulses from a dye laser (λ = 500 nm, τ = 3.5 ns, Ε ≈ 5 μJ) are applied concentrically to the cw-laser spot on the eyeground. The absorption of the pulses lead to a consequent heating and thermoelastic expansion of the tissue. This causes the emission of an ultrasonic pressure wave, which amplitude was found to be temperature dependent following in good approximation a 2nd order polynomial. The pressure wave was measured by an ultrasonic transducer embedded in a contact lens placed on the cornea. The experiments were performed in-vivo on rabbits. Simultaneous measurements with a miniaturized thermocouple showed a similar slope with a maximum local deviation of 0.4 °C for a temperature increase of 5.5 °C. On two rabbits measurements pre and post mortem at the same location were performed. The temperature increase after 60 s was found to raise by 12.0 % and 66.7 % post mortem, respectively.
These data were used to calculate the influence of heat convection by blood circulation using a numerical model based on two absorbing layers and assuming a constant perfusion rate for the choriocapillaris and the choroid.
Overall the presented optoacoustic method seems feasible for a non-invasive real-time determination of cw-laser induced retinal temperature increases and might serve as a temperature based dosimetry control during retinal laser treatments.
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Rapid development of new laser technologies enabled the application of ultra short lasers in refractive surgery. Focused ultra short laser pulses in near-infrared spectral range can generate a laser induced breakdown (LIB) in the cornea, which will disrupt the tissue. Cutting depth and position can be established by varying the laser focus. The fs-LASIK technique allows both flap and lenticule to be formed by using fs-pulses without the presence of any mechanical impact. During the cutting process not all of the pulse energy is deposited into the cornea; approximately half of the remaining energy propagates through the eye and reaches the retina. Though defocused, the transmitted energy can still induce damage to the retina due to absorption by the retinal pigment epithelium and the transfer of thermal energy to surrounding tissue. The fs-LASIK process was simulated with two laser systems; one continous-wave and one in the fs-regime. For the simulation the exposure time and focusing numerical aperature which defines the retinal spot size were varied. The Damage thresholds of the laser beam exposed eyes were determined in terms of ophthalmoscopic and histopathologic observations.
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Influence of laser parameters are studied and discussed in this paper dealing with safety aspects of femtosecond laser refractive surgery. Studies on transmission and energy have been done using different focusing objectives. More than 20% of NIR photons are reaching the retina using a numerical aperture of 0.3 which represent a fluence of 2 μJ/cm2 with parameters for flap procedure of about 7 μJ energy per pulse, at 10 KHz repetition rate and at a wavelength of 1040 nm. When using an objective with NA 0.9 about one order less fluence will strike the retina. To minimize bubbles expansion and side effects in tissue processing as well as to exclude any damages at retina, a compromise between ablation time, pulse energy and process strategy by the use of higher numerical aperture has to be taken into account.
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Human retina consists of multiple layers, with oxygen supply from chorioidal and retinal vascular circulations. A number of ocular disorders are associated with insufficient oxygen supply in the retinal layer. However no effective method has been developed yet to quantify the retinal tissue oxygen saturation. Diffuse optical imaging and spectroscopy (DOIS) offers a new opportunity for tissue oximetry. The technique is non-invasive, low cost, non-radioactive and real time. However, the application of DOIS in ocular imaging is hindered by the following limitations: 1) lack of spatial and depth resolution; 2) light transportation in thin layers less than single mean free path; 3) low scattering coefficient in neural retina and high absorption coefficient in RPE; 4) interference by retinal vessels. This paper discussed both theoretical and experimental works toward quantitative assessment of retinal tissue oxygenation. Theoretical side, photon migration in multi-layer tissue was simulated by solving diffusion equations in Fourier domain. The resulting diffuse reflectance was compared with Monte Carlo simulation. Experimental side, a dual modal imaging prototype was developed combining white light interferometry for tissue thickness measurement and near infrared spectroscopy for optical property measurement. The capability for white light interfferometry to capture thin layer thickness was demonstrated by a series of benchtop tests.
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Adaptive Optics assisted eye fundus imaging now provides high resolution images of the very contents of the retinal tissue. Impressive images of the mosaic of the photoreceptors have obtained by several groups and the best reported images even show the circulatory activity in capillars. Nevertheless, the natural eye movements, such as drifts or micro-saccades, generally prevent from an accurate registration of the field of view. Moreover, images obtained through scanning techniques also suffer from field distorsion. These two effects make it difficult to unambiguously extract blood cell density and velocity information. We report how Adaptive Optics assisted flood imaging of the retina, associated with an original a posteriori image registration and data analysis now permits evaluating and mapping the flow activity in the retinal micro-vessels. Examples where capillars as thin as a few micrometers are revealed only by their circulatory activity will be given. Larger field of view along the retinal vascular network will also be produced. Thanks to this high resolution functional mapping, we'll demonstrate how blood cells direct observation can be enhanced and how quantitative parameters such as velocity or density can be derived. Velocity maps of the macular vascular network at the eye's diffraction limit will finally be produced.
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A fundus camera was modified to illuminate the retina of a rabbit model with low power laser light in order to obtain laser speckle images. A fast-exposure charge-coupled device (CCD) camera was used to capture laser speckle images of the retina. Image acquisition was synchronized with the arterial pulses of the rabbit to ensure that all images are obtained at the same point in the cardiac cycle. The rabbits were sedated and a speculum was inserted to prevent the eyelid from closing. Both albino (New Zealand) and pigmented (Dutch belted) rabbits were used in the study. The rabbit retina is almost avascular. The measurements are obtained for choroidal tissue as well as retinal tissue. Because the retina is in a region of high metabolism, blood velocity is strongly affected by blood oxygen saturation. Measurements of blood velocity obtained over a wide range of O2 saturations (58%-100%) showed that blood velocity increases with decreasing O2 saturation. For most experiments, the left eye of the rabbit was used for laser measurements whereas the right eye served as a control. No observable difference between pre- and post-experimented eye was noted. Histological examinations of retinal tissue subjected to repeated laser measurements showed no indication of tissue damage.
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Two-dimensional distribution of oxygen saturation levels across the human retina is predicted by measuring the multispectral images. A liquid crystal tunable filter is used to scan wavelength ranging from 500 to 700 nm to acquire the multispectral images with the spectral resolution of 20 nm. After preprocessing reflected spectra at every pixel to cancel common offsets and amplifications, the partial least squares regression is adopted to estimate the oxygen saturation levels at each pixel point. All the resulted response values compose the oxygen saturation level map. The effects of preprocessing types on the predicted oxygen saturation levels are also discussed.
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An optical system designed for exposure of rabbit eyes to laser radiation and in-situ retinal damage assessment is presented. The laser radiation is of 2nd harmonic Q-switched Nd:YAG laser at 532 nm. The system is designed for multiple exposures at a regular grid array within a pre-determined region of the retina. Damage assessment is done in real time parallel to the exposure process. We present experimental results that demonstrate the versatility of the system for the determination of the threshold for laser-induced retinal damage in rabbit eye.
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Laser welding of corneal tissue is an alternative technique to conventional suturing procedures in ophthalmic surgery. The welding effect is achieved after staining the wound with a chromophore (Indocyanine Green, shortly: ICG) and then irradiating it with a low power diode laser. We present a study on the healing process of corneal wounds using Multispectral Imaging Autofluorescence Microscopy (MIAM). This technique is based on the characterization of fluorescence arising from tissue components (autofluorescence): it is particularly useful in studying corneal tissue, because it is mainly composed of type I collagen, one of the most important endogenous fluorophores. Laser welding tests of the cornea were carried out on rabbits in which full thickness corneal cuts of about 5 mm were sutured using a diode laser emitting at 810 nm, with a power of 80 mW. Bioptic sections of rabbit corneas were examined in a follow up study of 90 days after surgery, and the results were complementary to histological analysis performed in previous studies. Autofluorescence images showed a faster healing process and a better reorganization of the architecture of stromal fibers, in comparison with conventional suturing procedures. MIAM technique can represent a new tool to study the morphology of corneal tissue, offering some real advantages with respect to standard histological analysis. In fact, it does not require any chemical manipulation of the samples, providing information on the biological structure by directly monitoring distribution and emission intensity of endogenous fluorophores.
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Purpose: To demonstrate the biocompatibility of SIBS implants as compared to PDMS implants in the treatment of retinal detachment in New Zealand White (NZW) rabbit model.1,2Introduction: Scleral encircling bands, fixation rings and buckles are utilized for closure of retinal breaks and retina reattachment. The FDA approved PDMS-implant is associated with several post-operative complications, involving thick-fibrotic encapsulations. SIBS, an elastomeric triblock copolymer, was recently FDA approved for use in a cardiovascular drug eluting stent (TAXUSTM, Boston Scientific Corp., MA) and showed excellent biocompatibility and slow drug release capability. Materials and Methods: SIBS (9-mol%-styrene) implants were fabricated (InnFocus LLC, USA) to match PDMS implants (Labtician, Inc, Canada) dimensions. 5 NZW rabbits received SIBS and 4, PDMS-implants. Post-operative exam sequence: day 1 and 2, week 1, 2, 3, 4 and 6, and monthly thereafter for up to 1 year. Anatomohistopathology exams sequence: one SIBS animal at 6 weeks and one animal of each treatment group at 3 and 6-months, and two at 12-months. Results: SIBS compared to PDMS animals exhibited less inflammation and a better buckling effect during the first 6 weeks. At POD 9 months, the conjunctival injection in the SIBS rabbit was none as opposed to the PDMS value and the buckling effect for both groups were equal. There were no visible signs of encapsulation with SIBS. There were no infections in the 9 animals and none of the implants extruded thus far (<10 months). Conclusion: SIBS encircling bands, sleeves, and buckle implants are well tolerated in the rabbit model.
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The purpose of this investigation is to characterize corneal wound healing under in vitro conditions. Multiphoton
autofluorescence and second harmonic generation (SHG) microscopy will be used to visualize cells and collagen fibers
associated with corneal wound healing. Using the near-infrared excitation source from a titanium-sapphire laser pumped
by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton
autofluorescence and SHG signals from the cornea specimens. A home-modified commercial microscope system with
specified optical components is used for optimal signal detection. To acquire both high resolution and tissue-level
information of the specimen, a sample positioning stage is used in conjunction with the beam scanning system. Finally,
the organ level image can be assembled from individual area scans. The in vitro samples we used are cornea buttons
acquired from porcine eyes. Localized wounds will be induced by #11 blade and imaged using multiphoton microscopy.
Based on these results, we envision the in vitro imaging chamber to be able to follow the wound healing process without
damaging histological procedures. We envision this approach will enable us to further understand wound healing process
associated with corneal scar and can lead to in vivo methodology for diagnosing cornea damage.
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The purpose of this study is to assess the application of multiphoton fluorescence and second harmonic generation (SHG)
microscopy for imaging and monitoring the disease progress of infectious keratitis in an experimental model, and to
investigate the possible correlation of tissue architecture with spreading patterns of pathogens in an experimental model.
Porcine eyes are to be obtained from slaughter house and processed and placed in organ culture system. Fungal
infections by common pathogens of infectious keratitis are to be induced in porcine cornea buttons. Multiphoton
fluorescence and SHG microscopy will be used for imaging and for monitoring the progression and extension of tissue
destruction and possibly the pattern of pathogen spreading. We found that SHG imaging is useful in identifying
alterations to collagen architecture while autofluorescence microscopy can be used to visualize the fungi and cells within
the stroma. In summary, multiphoton fluorescence and second harmonic generation microscopy can non-invasively
demonstrate and monitor tissue destruction associated with infectious keratitis. The pattern of pathogen spreading and its
correlation with the tissue architecture can also be shown, which can be useful for future studies of the tissue-microbial
interactions for infectious keratitis.
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Lid angles and aperture size are important factors for fitting and on-eye performance of contact lenses. In particular, toric and translating bifocal lenses rely on predictable interaction with the eyelid for successful positioning and orientation. Traditionally, lid assessment and fitting evaluation is performed under slit lamp observation. Observations are limited to primary gaze and nasal or temporal directions. Quantification of lid parameters and lens positioning is subjective and depended on the skill of the practitioner. No commercial instrument is available off the shelf, which would be suitable to measure objectively ocular parameters and/or contact lens positioning under different directions of gaze. This is the likely reason why only one study has been published in which ocular characteristics for different gaze angles were obtained [1]. However, only a limited number of parameters and gaze directions were investigated. Almost all contact lenses are designed for and assessed under primary gaze. Considering that patients constantly change their gaze while wearing contact lenses, the quantification of contact lens movement and changes in ocular parameters with eye movement can contribute substantially to the understanding of lens performance and thereby lead to improved lens designs. This is of particular importance for toric and translating bifocal lenses. Their complex optical and topographical design requires precise positioning, orientation and movement with changes in gaze direction in order to provide adequate vision. Baron [2] suggested that the lower eyelid is the most important factor for lens movement of translating bifocal lenses, but questions still remained on the complete dynamics of on eye behaviour. For toric contact lenses, the rotational orientation is more important than vertical translation to obtain optimum visual correction. Most toric lenses feature a prism ballast design. Blinking movements, mainly of the upper eyelid, interact with the wedge like shape and squeeze the thick part downwards. It has been observed that the upper eyelid does not move in a straight downward movement, but also twists slightly to close the temporal side first [3]. Precise quantification of this motion and how it affects the contact lens movement have not been reported. A new instrument was designed and constructed to provide objective measurements of ocular parameters.
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Additional Paper from Session Retinal Imaging III: Adaptive Optics
Adaptive Optics - Optical Coherence Tomography (AO-OCT) has demonstrated a promising improvement in lateral
resolution for retinal imaging compared to standard OCT. Recent developments in Fourier-domain OCT technology
allow AO-OCT instruments to acquire three-dimensional (3D) retinal structures with high speed and high "volumetric"
resolution (in all three dimensions). One of the most important factors (besides acquisition speed) that will determine the
true potential of this technique is its ability to achieve diffraction-limited lateral resolution (~3 μm) while operating in
the ultrahigh axial resolution range (~3 μm) offered by OCT. Theoretical studies have shown that the eye's chromatic
aberrations may drastically reduce volumetric resolution. This is a critical finding because for "standard" stand alone
ultrahigh OCT, increasing the spectral bandwidth of the light source improves axial resolution without compromising
lateral resolution. To study the effects of spectral bandwidth on AO-OCT systems for retinal imaging two different light
sources offering 6 and 3 μm axial resolution were tested. This comparison was based on both AO correcting system
performance as well as the quality of corresponding OCT images.
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