This PDF file contains the editorial “Editorial: JBO Begins Bimonthly Publication” for JBO Vol. 9 Issue 01

This PDF file contains the editorial “Guest Editorial: Ophthalmic Diagnostics” for JBO Vol. 9 Issue 01

An overview of the fluorophores of the eye, the methods used to measure ocular fluorescence, and the existing or ready-to-market instrumentation for the early diagnosis and monitoring of ophthalmic pathologies is presented. Emphasis is given to the impact that a more detailed knowledge of the fluorophores of the eye, their age and pathology relationship, and the considerable technology-driven progress in optical devices and components has had on the development of new compact yet powerful instruments for population screening and for patient follow-ups, using fluorometry alone or in combination with other optical techniques such as light scattering.

The noninvasive techniques of static and dynamic light scattering are emerging as valuable diagnostic tools for the early detection of ocular and systemic diseases. These include corneal abnormalities, pigmentary dispersion syndrome, glaucoma, cataract, diabetic vitreopathy, and possibly macular degeneration. Systemic conditions such as diabetes and possibly Alzheimer's disease can potentially be detected early via ocular tissues. The current state of development of these techniques for application to ophthalmic research and ultimately clinical practice is reviewed.

Imaging the vitreous is an attempt to view what is by design invisible. The inability to adequately image vitreous hinders a more complete understanding of its normal structure and function and how these change in aging and disease. The combined use of more than one technique could provide better imaging for investigational and clinical purposes. Past and present imaging methodologies are summarized and research and clinical techniques that are currently in development for future applications, are discussed. Dark-field slit microscopy has been used to characterize vitreous anatomy, both within the vitreous body as well as at the vitreo–retinal interface. In addition to this methodology, slit-lamp biomicroscopy; direct, indirect, and scanning laser ophthalmoscopies; ultrasonography; optical coherence tomography; magnetic resonance and Raman spectroscopies; and dynamic light-scattering methodologies for noninvasive evaluation are presented. Dark-field slit microscopy enables in vitro imaging without dehydration or tissue fixatives. Optical coherence tomography enables better in vivo visualization of the vitreo-retinal interface than scanning laser ophthalmoscopy and ultrasonography, but does not image the vitreous body. Dynamic light scattering can determine the average sizes of vitreous macromolecules within the vitreous body as well as possibly image the posterior vitreous cortex once detached, while Raman spectroscopy can detect altered vitreous molecules, such as glycated collagen and other proteins in diabetic vitreopathy.

In the past two decades, optical coherence tomography (OCT) has been established as an adjunct diagnostic technique for noninvasive, high-resolution, cross-sectional imaging in a variety of medical fields. The rapid development of ultrabroad bandwidth light sources has recently enabled a significant improvement in OCT imaging resolution, demonstrating the potential of OCT to accomplish its original goal of performing noninvasive optical biopsies, i.e., the in vivo visualization of microstructural morphology in situ, which had previously only been possible with histopathology. In addition, these novel light sources might also enable the use of spectroscopic OCT, an extension of ultrahigh-resolution OCT, for enhancing image contrast as well as detecting spatially resolved functional, biochemical tissue information. State-of-the-art-light sources that now permit ultrahigh-resolution OCT covering the whole wavelength region from 500 to 1600 nm are reviewed and fundamental limitations of OCT image resolution are discussed. Ex vivo ultrahigh-resolution OCT tomograms are compared with histological results; first clinical in vivo ultrahigh-resolution OCT and preliminary spectroscopic OCT results are presented and their impact for future clinical and research applications is discussed.

There is currently strong interest in developing noninvasive technologies for the detection of macular carotenoid pigments in the human eye. These pigments, consisting of lutein and zeaxanthin, are taken up from the diet and are thought to play an important role in the prevention of age-related macular degeneration, the leading cause of blindness in the elderly in the Western world. It may be possible to prevent or delay the onset of this debilitating disease with suitable dietary intervention strategies. We review the most commonly used detection techniques based on heterochromatic flicker photometry, fundus reflectometry, and autofluorescense techniques and put them in perspective with recently developed more molecule-specific Raman detection methods.

We demonstrate the clinical application of a multiplanar imaging system that simultaneously acquires en face (C-scan) optical coherence tomography (OCT) and the corresponding confocal ophthalmoscopic images, along with cross-sectional (B-scan) OCT at specifiable locations on the confocal image. The advantages of the simultaneous OCT and confocal acquisition as well as the challenges of interpreting the C-scan OCT images are discussed. Variations in tissue inclination with respect to the coherence wave surface alter the sampling of structures within the depth of the retina, producing novel slice orientations that are often challenging to interpret. We have evaluated for the first time the utility of C-scan OCT for a variety of pathologies, including melanocytoma, diabetic retinopathy, choroidal neovascular membrane, and macular pucker. Several remarkable new aspects of clinical anatomy were revealed using this new technique. The versatility of selective capture of C-scan OCT images and B-scan OCT images at precise points on the confocal image affords the clinician a more complete and interactive tool for 3-D imaging of retinal pathology.

Optical coherence tomography (OCT) is an emerging technology for high-resolution, noncontact imaging of transparent and scattering media. Polarization-sensitive optical coherence tomography (PS-OCT) is a functional extension of OCT that can image birefringent properties of a biological sample. PS-OCT was used to measure and image retardation and birefringent axis orientation of in vitro human cornea. We used a two-channel PS-OCT system employing a phase-sensitive recording of the interferometric signals in two orthogonal polarization channels. Using an algorithm based on a Hilbert transform, it is possible to calculate the retardation and the slow axis orientation of the sample with only a single A-scan per transversal measurement location. While the retardation information is encoded in the amplitude ratio of the two interferometric signals, the axis orientation is encoded entirely in their phase difference. We present maps of retardation and the distribution of slow axis orientation of the human cornea in longitudinal cross-sections and en face images obtained at the back surface of the cornea. The retardation increases in a radial direction and with depth; the slow axis varies in the transversal direction. Knowledge of the retardation and the slow axis distribution of the cornea might improve nerve fiber polarimetry for glaucoma diagnostics and could be useful for diagnosing different types of pathologies of the cornea.

A new optical scheme to detect glucose concentration in the aqueous humor of the eye is presented. The ultimate aim is to apply this technique in designing a new instrument for, routinely and frequently, noninvasively monitoring blood glucose levels in diabetic patients without contact (no index matching) between the eye and the instrument. The optical scheme exploits the Brewster reflection of circularly polarized light off of the lens of the eye. Theoretically, this reflected linearly polarized light on its way to the detector is expected to rotate its state of polarization, owing to the presence of glucose molecules in the aqueous humor of a patient's eye. An experimental laboratory setup based on this scheme was designed and tested by measuring a range of known concentrations of glucose solutions dissolved in water.

A rapid, sensitive, and cost-effective method is reported for the subjective and objective documentation of subtle opacities in lenses of unanesthetized transgenic mice or selenite-injected rat pups as models for cataract formation. Animal eyes were dilated with eye drops and the animal was positioned in front of a Nikon FS2 photo slit lamp. Slit-lamp observations were recorded using a Canon Optura Pi digital video recorder. High-quality images of opacifying lenses were captured from the video and quantified using densitometry at progressive stages of opacification. In mice, targeted genomic deletion of the proteins CP49 (a lens-specific filament) or Six5 (a model for myotonic dystrophy) resulted in subtle cataracts that were easily recorded and quantified using this instrumentation. In rats, the early progressive changes leading to a dense nuclear opacity caused by selenite injection were easily documented using this instrumentation. Low-cost components combined with a conventional slit-lamp ophthalmoscope were used to capture high-quality images of selected stages of cataract formation for quantitative analysis using commercial software.

Glaucoma causes damage of the nerve fiber layer, which may cause loss of retinal birefringence. Therefore, PS-OCT is a potentially useful technique for the early detection of glaucoma. We built a fiber-based PS-OCT setup that produces real-time images of the human retina in vivo, coregistered with retinal video images of the location of PS-OCT scans. Preliminary measurements of a healthy volunteer show that the double-pass phase retardation per unit of depth of the RNFL is not constant and varies with location, with values between 0.18 and 0.37 deg/µm. A trend in the preliminary measurements shows that the nerve fiber layer located inferior and superior to the optic nerve head is more birefringent than the thinner layer of nerve fiber tissue in the temporal and nasal regions.

Retinopathy of prematurity (ROP) describes a pathological development of the retina in prematurely born children. In order to prevent severe permanent damage to the eye and enable timely treatment, the fundus of the eye in such children has to be examined according to established procedures. For these examinations, our miniaturized fundus camera is intended to allow the acquisition of wide-angle digital pictures of the fundus for on-line or off-line diagnosis and documentation. We designed two prototypes of a miniaturized fundus camera, one with graded refractive index (GRIN)-based optics, the other with conventional optics. Two different modes of illumination were compared: transscleral and transpupillary. In both systems, the size and weight of the camera were minimized. The prototypes were tested on young rabbits. The experiments led to the conclusion that the combination of conventional optics with transpupillary illumination yields the best results in terms of overall image quality.

We present axial resolution calculated using a mathematical model of the adaptive optics scanning laser ophthalmoscope (AOSLO). The peak intensity and the width of the axial intensity response are computed with the residual Zernike coefficients after the aberrations are corrected using adaptive optics for eight subjects and compared with the axial resolution of a diffraction-limited eye. The AOSLO currently uses a confocal pinhole that is 80 µm, or 3.48 times the width of the Airy disk radius of the collection optics, and projects to 7.41 µm on the retina. For this pinhole, the axial resolution of a diffraction-limited system is 114 µm and the computed axial resolution varies between 120 and 146 µm for the human subjects included in this study. The results of this analysis indicate that to improve axial resolution, it is best to reduce the pinhole size. The resulting reduction in detected light may demand, however, a more sophisticated adaptive optics system. The study also shows that imaging systems with large pinholes are relatively insensitive to misalignment in the lateral positioning of the confocal pinhole. However, when small pinholes are used to maximize resolution, alignment becomes critical.

Clinical studies of carotenoid macular pigments (MP) have been limited by the lack of noninvasive, objective instruments. We introduce a novel noninvasive optical instrument, an MP Raman detector, for assessment of the carotenoid status of the human retina in vivo. The instrument uses resonant excitation of carotenoid molecules in the visible wavelength range, and quantitatively measures the highly specific Raman signals that originate from the single- and double-bond stretch vibrations of the π-conjugated carotenoid molecule's carbon backbone. The instrument is a robust, compact device and suitable for routine measurements of MP concentrations in a clinical setting. We characterized and tested the instrument in clinical studies of human subjects to validate its function and to begin to establish its role as a possible screening test for macular pathologies. We also show that the MP Raman spectroscopy technology has potential as a novel, highly specific method for rapid screening of carotenoid antioxidant levels in large populations at risk for vision loss from age-related macular degeneration, the leading cause of blindness of the elderly in the developed world.

We describe a novel method for testing a visual field that employs a computer monitor with displays of varying contrast that permits unprecedented resolution and characterization of the structure of scotomas in three dimensions. Patients are placed in front of a touch-sensitive computer screen at a fixed distance. With one eye covered, they focus on a central fixation marker and trace with their finger the areas on an Amsler grid that are missing from their field of vision. Increasing degrees of contrast of the Amsler grid are simulated by repeating the test at different gray-scale levels. The results are recorded and then displayed as topographical contour rings by the computer test program. The results can also be rendered as an immediate 3-D depiction of the central hill-of-vision. Several clinical pilot studies have been conducted at the Doheny Eye Institute and more than 200 patients have been examined with this system so far. Conditions such as optic neuritis, anterior ischemic optic neuropathy (AION), age-related macular degeneration (AMD), glaucoma, and ocular hypertension have been successfully assessed by this test. Each condition provides unique patterns that are most evident in 3-D. The 3-D computer-automated threshold Amsler grid test is an innovative and noninvasive visual field test. It provides several advantages over state-of-the-art standard automated perimetry, including: (1) additional information through 3-D depiction of scotomas, such as location, extent, slope, depth, and shape; (2) high angular resolution (1 deg compared with typically 6 deg); (3) a simple test setup (merely a touch-sensitive computer monitor and the test software); (4) excellent patient compliance (spending 4 to 5 min per eye). In light of its promising initial tests, the 3-D visual field test appears to have the potential for the early detection and monitoring of various diseases over time.

The single-pass modulation transfer function (MTF_sgl) is an important numerical parameter that can help elucidate the performance and some processes of the human visual system. In previous studies, the MTF_sgl was calculated from double-pass point spread function (PSF) measurements. These measurements include a depolarized reflection component from the retina that introduces a measurement artifact, and they require long acquisition times to allow averaging to reduce speckle. To solve these problems, we developed a new ocular PSF analysis system (PSFAS) that uses polarization optics to eliminate the depolarized retinal reflection component, and a rotating prism to increase measurement speed. Validation experiments on one patient showed that the MTF_sgl measured by PSFAS agrees closely with the MTF calculated from contrast sensitivity measurements. A simulated retinal image was calculated by convolution of Landolt rings with the calculated single-pass PSF provided by the PSFAS. The contrast characteristic then was calculated from the simulated retinal images. These results indicate that the MTF_sgl obtained using the PSFAS may be a reliable measure of visual performance of the optics of the eye, including the optical effects of the retina. The simulated retinal images and contrast characteristics are useful for evaluating visual performance.

Normal macular photographic patterns are geometrically described and mathematically modeled. Forty normal color fundus photographs were digitized. The green channel gray-level data were filtered and contrast enhanced, then analyzed for concentricity, convexity, and radial resolution. The foveal data for five images were fit with elliptic quadratic polynomials in two zones: a central ellipse and a surrounding annulus. The ability of the model to reconstruct the entire foveal data from selected pixel values was tested. The gray-level patterns were nested sets of concentric ellipses. Gray levels increased radially, with retinal vessels changing the patterns to star shaped in the peripheral fovea. The elliptic polynomial model could fit a high-resolution green channel foveal image with mean absolute errors of 6.1% of the gray-level range. Foveal images were reconstructed from small numbers of selected pixel values with mean errors of 7.2%. Digital analysis of normal fundus photographs shows finely resolved concentric elliptical foveal and star-shaped parafoveal patterns, which are consistent with anatomical structures. A two-zone elliptic quadratic polynomial model can approximate foveal data, and can also reconstruct it from small subsets, allowing improved macular image analysis.

In all fundus laser treatments of the eye, the temperature increase is not exactly known. In order to optimize treatments, an online temperature determination is preferable. We investigated a noninvasive optoacoustic method to monitor the fundus temperature during pulsed laser irradiation. When laser pulses are applied to the fundus, thermoelastic pressure waves are emitted, due to thermal expansion of the heated tissue. Using a constant pulse energy, the amplitude of the pressure wave increases linearly with an increase in the base temperature of between 30 and 80°C. This method was evaluated in vitro on porcine retinal pigment epithelium (RPE) cell samples and clinically during selective RPE treatment with repetitive microsecond laser pulses. During the irradiation of porcine RPE with a neodymium-doped yttrium (Nd:YLF) laser (527 nm, 1.7 µs, 500 Hz repetition rate, 160 mJ/cm²) an increase in the base temperature of 30±4°C after 100 pulses was found. During patient treatments, a temperature increase of 60±11°C after 100 pulses with a 500-Hz repetition rate and 7±1°C after 30 pulses with 100 Hz at 520 mJ/cm2 was found. All measured data were in good agreement with heat diffusion calculations. Optoacoustic methods can be used to noninvasively determine retinal temperatures during pulsed laser treatment of the eye. This technique can also be adapted to continuous-wave photocoagulation, photodynamic therapy and transpupillary thermotherapy, or other fields of laser-heated tissue.

An aglorithm based on support vector machines (SVM), the most recent advance in pattern recognition, is presented for use in classifying light-induced autofluorescence collected from cancerous and normal tissues. The in vivo autofluorescence spectra used for development and evaluation of SVM diagnostic algorithms were measured from 85 nasopharyngeal carcinoma (NPC) lesions and 131 normal tissue sites from 59 subjects during routine nasal endoscopy. Leave-one-out cross-validation was used to evaluate the performance of the algorithms. An overall diagnostic accuracy of 96%, a sensitivity of 94%, and a specificity of 97% for discriminating nasopharyngeal carcinomas from normal tissues were achieved using a linear SVM algorithm. A diagnostic accuracy of 98%, a sensitivity of 95%, and a specificity of 99% for detecting NPC were achieved with a nonlinear SVM algorithm. In a comparison with previously developed algorithms using the same dataset and the principal component analysis (PCA) technique, the SVM algorithms produced better diagnostic accuracy in all instances. In addition, we investigated a method combining PCA and SVM techniques for reducing the complexity of the SVM algorithms.

We immunohistochemically studied expressions of inducible heat shock protein 70 (HSP 72) and apoptosis of corneas ablated with an ArF excimer laser. The temperature of corneal surfaces and laser-induced optical emission spectra were measured in real time as direct physical parameters related to the ablation mechanism. To the best of the authors' knowledge, there have been no experimental studies regarding the influence of physical parameters directly related to the ablation mechanism on corneal reactions at the cell level after laser ablation. The expression of HSP 72 was mainly localized in the regenerative epithelium, which was confirmed to be caused by laser ablation. The HSP 72 positive cell ratios had a correlation with thermal dose, which was derived from the measured time courses of temperature. Expressions of both HSP 72 and apoptosis depended on the thermal dose and elapsed time after ablation. HSP 72 and apoptosis could be seen up to a few hundred micrometers into the stroma, only at a fluence with an optical breakdown emission. This could have been caused by shock waves induced by the optical breakdown.

The effectiveness of an optical coherence tomography (OCT) system depends largely on the light source chosen. Published data on the optical properties of tissues are used to quantify the exponential attenuation of broadband light on transport through tissue. The effective attenuation coefficient is taken to be the sum of the absorption and scattering coefficients. This is used to demonstrate the effect on the spectra of a wide range of published OCT sources and the change in system resolution induced, and hence to comment on the suitability of different sources for OCT. The tissues studied include skin dermis, liver, and gallbladder. Sources at higher wavelengths are shown to be capable of high-resolution OCT imaging at greater depths. Titanium:sapphire lasers would be most suited for high-resolution OCT over comparatively shallow depths into tissue. For lower-resolution applications of OCT, a semiconductor optical amplifier and ytterbium fiber sources have better powers and bandwidths than superluminescent diodes. The resolution of OCT systems is not reduced significantly with imaging depth.

The depth of light penetration into highly scattering tissues can be improved by the application of biocompatible and osmotically active chemical agents. We compare the dynamics of optical clearing of tissue by the topical application of glycerol and dimethyl sulfoxide (DMSO) using optical coherence tomography (OCT). It is demonstrated experimentally that both agents can largely improve the OCT imaging depth for porcine stomach tissue. During a period of approximately 20 to 30 min after the application of glycerol image contrast is also enhanced. This enhancement disappears over time. Such enhancement of image contrast is not observed with DMSO. Glycerol causes a higher degree of dehydration of the tissue than DMSO does. We suggest that these phenomena are caused by a two-stage diffusion of the chemicals. The first stage of diffusion is from the top tissue to the intercellular space, and the second is into the cell matrix. During the first stage, the imaging contrast could be improved by dehydration.

An assessment of burn depth is a key step in guiding the treatment of patients who have sustained thermal injuries. Polarization-sensitive optical coherence tomography (PS-OCT) might eventually provide the physician with a quantitative estimate of actual burn depth. Burns of various depths were induced by contacting rat skin with a brass rod preheated to 75 °C for 5, 15, or 30 s. Thermal injury denatured the collagen in the skin, and PS-OCT imaged the resulting reduction of birefringence through the depth-resolved changes in the polarization state of light propagated and reflected from the sample. Stokes vectors were calculated for each point in the PS-OCT images and the reduction in the rate of phase retardation between two orthogonal polarizations of light (deg/µm) was found to show a consistent trend with burn exposure time. PS-OCT is a noninvasive technique with potential to give the physician the information needed to formulate an optimal treatment plan for burn patients.

The use and advantages of balanced detection for making low-noise polarimetric measurements in turbid materials are demonstrated. The technique reduces the intensity noise originating from the laser and, in addition, makes possible a direct measurement of a component of the Stokes vector. When phase-locked detection is used with either amplitude or polarization modulation for polarimetric measurements in turbid media, one can obtain elements of the scattering matrix of very small magnitude. This methodology is used to measure optical activity and surviving linear polarization fractions in clear and turbid media containing glucose at physiologically relevant concentrations. The results are in agreement with Monte Carlo simulations of polarized light propagation in turbid media.

The basic parameters for physiological measurements provided by near-infrared spectroscopy are the local absorption and scattering coefficients. For the adult human head, they have been difficult to measure noninvasively because of the layered structure of the head. The results of measurements of absorption and reduced scattering coefficients through the forehead on 30 adult volunteers using a multidistance frequency domain method are reported. The optode separation distance ranged from 10 to 80 mm and measurements were recorded at 758 and 830 nm. The measured absorption and reduced scattering coefficients of the forehead were used to evaluate the hemoglobin content in the scalp and brain as well as cerebral oxygen saturation. We found that cerebral oxygenation was relatively narrowly distributed within the subject group (the standard deviation was about 3% for scalp and 6% for brain, respectively), whereas hemoglobin concentrations had a relatively broader distribution. We found that as the optode distance increased, the absorption coefficients increased and the scattering coefficients decreased, retrieving the optical values of scalp and brain for shorter and longer optode distances, respectively. We present the transition curves of the absorption and reduced scattering coefficients as functions of the optode distance. In order to verify the values for each layer, a comparison between the experimental data and a prediction based on the two-layer model of the adult head was carried out. The thicknesses of scalp and skull for the two-layer model were obtained by magnetic resonance imaging of a subject's head. The optical parameters obtained from the two-layer model agreed very well with those measured by the multidistance method.

Presurgical chemotherapy is widely used in the treatment of locally advanced breast cancer. Monitoring the response to therapy can improve survival and reduce morbidity. We employ a noninvasive, near-infrared method based on diffuse optical spectroscopy (DOS) to quantitatively monitor tumor response to neoadjuvant chemotherapy. DOS was used to monitor tumor response in one patient with locally advanced breast cancer throughout the course of her therapy. Measurements were performed prior to doxorubicin-cyclophosphamide therapy and at several time points over the course of three treatment cycles (68 days). Our results show strong tumor to normal (T/N) tissue contrast in total hemoglobin concentration (T/N=2.4), water fraction (T/N=6.9), tissue hemoglobin oxygen saturation, StO2 (T/N=0.9), and lipid fraction (T/N=0.7) prior to treatment. Over a 10-week period, the peak total hemoglobin and water dropped 56 and 67%, respectively. Lipid content nearly returned to baseline (T/N = 0.9) while StO2 exceeded pretreatment levels (T/N =1.5). Approximately half of the hemoglobin and water changes occurred within 5 days of treatment (26 and 37%, respectively). These data suggest that noninvasive, quantitative optical methods that characterize tumor physiology may be useful in assessing and optimizing individual response to neoadjuvant chemotherapy.