Temporal change of retinal nerve fiber layer reflectance speckle in normal and hypertensive retinas.

This study investigated temporal change of retinal nerve fiber layer (RNFL) reflectance speckle in retinas with ocular hypertensive (OHT) damage and in control retinas from untreated eyes. Experimental OHT damage to rat retinas was induced by laser photocoagulation of the trabecular meshwork. A series of 660 nm reflectance images was collected from isolated retinas at 10-sec intervals. Areas containing speckled texture were selected on nerve fiber bundles. Correlation coefficients between images with different imaging delays were calculated and plotted as a function of delay. To evaluate the temporal change of speckles, decay of correlation coefficients with time was fitted with an exponential function characterized by a time constant τ. Reflectance per unit thickness (σ) of the areas was also measured and low σ was used as a surrogate of OHT damage. Speckle phenomena occurred in the control RNFL and the RNFL with reduced σ. In the control retinas, τ and σ were nearly constant along bundles but differed significantly among bundles in the same retinas. Among the control retinas, σ was similar, whereas τ varied significantly. In the retinas with OHT damage (low σ) τ could be within, greater or lower than the range in controls. The parameters τ and σ provide independent assessment of the RNFL with OHT damage. Measurements of temporal change of RNFL reflectance speckle may offer a method for detecting functional abnormality of the RNFL.

Registration of OCT fundus images with color fundus photographs based on blood vessel ridges.

This paper proposes an algorithm to register OCT fundus images (OFIs) with color fundus photographs (CFPs). This makes it possible to correlate retinal features across the different imaging modalities. Blood vessel ridges are taken as features for registration. A specially defined distance, incorporating information of normal direction of blood vessel ridge pixels, is designed to calculate the distance between each pair of pixels to be matched in the pair image. Based on this distance a similarity function between the pair image is defined. Brute force search is used for a coarse registration and then an Iterative Closest Point (ICP) algorithm for a more accurate registration. The registration algorithm was tested on a sample set containing images of both normal eyes and eyes with pathologies. Three transformation models (similarity, affine and quadratic models) were tested on all image pairs respectively. The experimental results showed that the registration algorithm worked well. The average root mean square errors for the affine model are 31 µm (normal) and 59 µm (eyes with disease). The proposed algorithm can be easily adapted to registration for other modality retinal images.

Dual-band spectral-domain optical coherence tomography for in vivo imaging the spectral contrasts of the retinal nerve fiber layer.

The ultimate goal of the study is to provide an imaging tool to detect the earliest signs of glaucoma before clinically visible damage occurs to the retinal nerve fiber layer (RNFL). Studies have shown that the optical reflectance of the damaged RNFL at short wavelength (<560 nm) is reduced much more than that at long wavelength, which provides spectral contrast for imaging the earliest damage to the RNFL. To image the spectral contrast we built a dual-band spectral-domain optical coherence tomography (SD-OCT) centered at 808 nm (NIR) and 415 nm (VIS). The light at the two bands was provided by the fundamental and frequency-doubled outputs of a broadband Ti:Sapphire laser. The depth resolution of the NIR and VIS OCT systems are 4.7 µm and 12.2 µm in the air, respectively. The system was applied to imaging the rat retina in vivo. Significantly different appearances between the OCT cross sectional images at the two bands were observed. The ratio of the light reflected from the RNFL over that reflected from the entire retina at the two bands were quantitatively compared. The experimental results showed that the dual-band OCT system is feasible for imaging the spectral contrasts of the RNFL.

Characteristics of optic nerve head drusen on optical coherence tomography images.

BACKGROUND AND OBJECTIVE: To describe the characteristics of optic nerve head drusen in optical coherence tomography (OCT) images. PATIENTS AND METHODS: Cross-sectional images of the optic nerve were obtained in seven patients with optic nerve head drusen with Stratus and spectral-domain OCT (Carl Zeiss Meditec, Dublin, CA). These were compared to optic disc photographs, autofluorescence, and echography images. For comparison, these tests were performed on four patients with papilledema and three patients with small optic discs. RESULTS: Optic nerve head drusen typically elevated the disc surface and appeared as an optically empty cavity, sometimes with a perceptible reflection from the posterior surface. The disc surface was also elevated in cases of papilledema, but had a strong anterior reflectance behind which there was no visible structure. The surface of the small optic nerves was slightly elevated, but with less anterior reflectance. CONCLUSION: Optic nerves with drusen showed features in these OCT images that were distinct from cases of papilledema or small optic discs.

Altered F-actin distribution in retinal nerve fiber layer of a rat model of glaucoma.

Glaucoma damages the retinal nerve fiber layer (RNFL). The purpose of this study was to investigate the distribution in RNFL of axonal F-actin, a cytoskeletal component, under the development of glaucoma. Intraocular hypertension was induced in a rat model by translimbal laser photocoagulation of the trabecular meshwork. The retinas of control and treated eyes were obtained after different exposures to elevated IOP. Nerve fiber bundles were identified by fluorescent phalloidin staining of F-actin. Nuclei of cell bodies were identified by DAPI fluorescent counterstain. F-actin distribution in whole-mounted retinas was examined by confocal microscopy. En face and cross-sectional images of RNFL were collected around the optic nerve head (ONH). F-actin in normal RNFL was intensely and uniformly stained. In glaucomatous retina, F-actin staining was not uniform within bundles and total loss of F-actin staining was found in severely damaged areas. Altered F-actin often occurred near the ONH in bundles that appeared normal more peripherally. Both alteration and total loss of F-actin were found most often in dorsal retina. In normal RNFL, F-actin is rich and approximately uniformly distributed within nerve fiber bundles. Elevated IOP changes F-actin distribution in RNFL. Topographic features of F-actin alteration suggest that F-actin near the ONH is more sensitive to glaucomatous damage. The alteration pattern also suggests an ONH location for the glaucomatous insult in this rat model.

Spectral domain optical coherence tomographic imaging of geographic atrophy.

BACKGROUND AND OBJECTIVE: To compare images of geographic atrophy (GA) obtained using spectral domain optical coherence tomography (SD-OCT) with images obtained using fundus autofluorescence (FAF). PATIENTS AND METHODS: Five eyes from patients with dry AMD were imaged using SD-OCT and FAF, and the size and shape of the GA were compared. RESULTS: GA appears bright on SD-OCT compared with the surrounding areas with an intact retinal pigment epithelium because of increased reflectivity from the underlying choroid. SD-OCT and FAF both identified GA reproducibly, and measurement of the area of GA is comparable between the two methods with a mean difference of 2.7% of the total area. CONCLUSION: SD-OCT can identify and quantitate areas of GA. The size and shape of these areas correlate well to the areas of GA seen on autofluorescence images; however, SD-OCT imaging also provides important cross-sectional anatomic information.

Corneal birefringence mapped by scanning laser polarimetry.

Corneal birefringence affects polarization-sensitive optical measurements of the eye. Recent literature supports the idea that corneal birefringence is biaxial, although with some disagreement among reports and without considering corneas with very low values of central retardance. This study measured corneal retardation in eyes with a wide range of central corneal retardance by means of scanning laser polarimetry (GDx-VCC, Carl Zeiss Meditec, Inc.), which computes the retardance and slow axis of the cornea from images of the bow tie pattern formed by the radial birefringence of the macula. Measurements were obtained at many points on the cornea by translating the instrument. Data were compared to calculations of the retardation produced by a curved biaxial material between two spherical surfaces. Most corneas showed one or two small areas of zero retardance where the refracted ray within the cornea aligned with an optical axis of the material. The retardation patterns in these corneas could be mimicked, but not accurately described, by the biaxial model. Two corneas with large areas of low retardance more closely resembled a uniaxial model. We conclude that the cornea, in general, behaves as a biaxial material with its fastest axis perpendicular to its surface. Some locations in a few corneas can be uniaxial with the optical axis perpendicular to the surface. Importantly, corneal birefringence varies greatly among people and, within a single cornea, significantly with position.

Three-dimensional spectral-domain optical coherence tomography images of the retina in the presence of epiretinal membranes.

PURPOSE: To study the inner surface of the retina in the presence of epiretinal membranes (ERMs) using a prototype spectral-domain optical coherence tomography (SD-OCT) device. DESIGN: Small case series, performed in the Department of Ophthalmology, Bascom Palmer Eye Institute, Miller School of Medicine, University of Miami, from August 2005 through December 2006. METHOD: An 8-microm axial-resolution SD-OCT instrument was used to scan the eyes of patients diagnosed with ERM. The ERM and the internal limiting membrane (ILM) were segmented separately to evaluate the traction caused by the ERM on the retina. It was then possible to reconstruct the ILM and ERM surfaces in 3-dimensional space and to obtain corresponding retinal thickness maps. RESULTS: SD-OCT B scans showed the points of attachment of the ERM to the ILM. Segmented surface maps of the ERM produced very smooth sheets, whereas those of the ILM presented wrinkles under and around the ERM. CONCLUSIONS: SD-OCT revealed the geometry of retinal traction in eyes with ERM and may be useful in understanding further the pathologic features of these lesions.

Macular thickness measurements in normal eyes using spectral domain optical coherence tomography.

BACKGROUND AND OBJECTIVE: Knowledge of the macular thickness in a normal population is important for the evaluation of pathological macular change. The purpose of this study was to define and measure macular thickness in normal eyes using spectral domain optical coherence tomography (OCT). PATIENTS AND METHODS: Fifty eyes from 50 normal subjects (29 men and 21 women, aged 22 to 68 years) were scanned with a prototype Cirrus HD-OCT system (5 microm axial resolution) (Carl Zeiss Meditec, Inc.). The proprietary Cirrus segmentation algorithm was used to produce retinal thickness maps, which were then averaged over 9 regions defined by a circular target centered at the true fovea location. The macular thickness of 13 subjects scanned with both HD-OCT and StratusOCT were compared. RESULTS: After centering the fovea, the mean and standard deviation values for retinal thickness measurements were calculated point wise and averaged on standard regions. For patients scanned with both systems, the thickness measurements from HD-OCT were approximately 50 microm larger than those from StratusOCT. The difference between the two measurements decreased somewhat with eccentricity. CONCLUSION: Using HD-OCT, it is possible to acquire retinal data sets containing an unprecedented number of data points. Furthermore, it is possible to use OCT fundus images to evaluate the scan quality and to center the measurement at the fovea. These advantages, together with good automated segmentation, can produce more accurate retinal thickness measurements. Incorporation of the photoreceptor layer in the measurements is anatomically meaningful and may be significant in evaluating various retinal pathologies and visual acuity outcomes.

Spectral domain optical coherence tomography for proliferative diabetic retinopathy with subhyaloid hemorrhage.

A prototype 6-microm axial resolution spectral domain optical coherence tomography (SD-OCT) device was used to image the retina of a patient with uncontrolled diabetes mellitus who had proliferative diabetic retinopathy with subhyaloid hemorrhage. A raster scan pattern with 128 B-scans covering a 6 X 6 X 2-mm volume of the retina was obtained. SD-OCT showed the presence of blood localized between the internal limiting membrane and the posterior hyaloid face and allowed visualization of the cross sectional retinal architecture and the vitreoretinal interface at different horizontal levels that could be registered with the color fundus photograph. SD-OCT provided useful information about the relationship of the hemorrhage to the posterior hyaloid and the retina.

Calibration of fundus images using spectral domain optical coherence tomography.

BACKGROUND AND OBJECTIVE: Measurements performed on fundus images using current software are not accurate. Accurate measurements can be obtained only by calibrating a fundus camera using measurements between fixed retinal landmarks, such as the dimensions of the optic nerve, or by relying on a calibrated model eye provided by a reading center. However, calibrated spectral domain OCT (SD-OCT) could offer a convenient alternative method for the calibration of any fundus image. PATIENTS AND METHODS: The ability to measure exact distances on SD-OCT fundus images was tested by measuring the distance between the center of the fovea and the optic nerve. Calibrated SD-OCT scans measuring 6 X 6 X 2 mm centered on the fovea and the optic nerve were analyzed in 50 healthy right eyes. The foveal center was identified using cross-sectional SD-OCT images, and the center of the optic nerve was identified manually. The SD-OCT scans were registered to each other, and the distances between the center of the optic nerve and fovea were calculated. The overlay of these SD-OCT fundus images on photographic fundus images was performed. RESULTS: Any image of the fundus could be calibrated by overlaying the SD-OCT fundus image, and the measurements were consistent with previously defined calibration methods. The mean distance between the center of the fovea and the center of the optic nerve was 4.32 +/-0.32 mm. The line from the center of the optic nerve to the foveal center had a mean declination of 7.67 +/- 3.88 degrees. Mean horizontal displacement and vertical displacement were 4.27 +/- 0.29 mm and 0.58 +/- 0.29 mm, respectively. CONCLUSIONS: The overlay of the SD-OCT fundus image provides a convenient method for calibrating any image of the fundus. This approach should provide a uniform standard when comparing images from different devices and from different reading centers.

Documentation of optic nerve pit with macular schisis-like cavity by spectral domain OCT.

The authors report using spectral domain optical coherence tomography (OCT) to observe a patient with an optic nerve pit and macular schisis-like spaces. An 8-microm axial resolution prototype spectral domain OCT and stereo fundus photography were used to observe the patient. A macular schisis-like cavity was present at baseline and additional cystic changes developed in the nerve fiber layer over a period of 16 months; however, the visual acuity remained stable at 20/20. Spectral domain OCT provides greater detail of the changes in morphology and structure of macular schisis and edema associated with an optic nerve pit.

Documentation by spectral domain OCT of spontaneous closure of idiopathic macular holes.

An observational case series using an 8-microm axial resolution prototype spectral domain optical coherence tomography (OCT) system was performed in two patients with idiopathic macular holes. Spontaneous closure and visual acuity improvement occurred in both patients. Useful information about morphology and vitreoretinal relationship of the holes was provided by spectral domain OCT.

Cytoskeletal Alteration and Change of Retinal Nerve Fiber Layer Birefringence in Hypertensive Retina.

PURPOSE: Glaucoma damages the retinal nerve fiber layer (RNFL). Both RNFL thickness and retardance can be used to assess the damage, but birefringence, the ratio of retardance to thickness, is a property of the tissue itself. This study investigated the relationship between axonal cytoskeleton and RNFL birefringence in retinas with hypertensive damage. MATERIALS AND METHODS: High intraocular pressure (IOP) was induced unilaterally in rat eyes. RNFL retardance in isolated retinas was measured. Cytostructural organization and bundle thickness were evaluated by confocal imaging of immunohistochemical staining of the cytoskeletal components: microtubules (MTs), F-actin, and neurofilaments. Bundles with different appearances of MT stain were studied, and their birefringence was calculated at different radii from the optic nerve head (ONH) center. RESULTS: Forty bundles in eight normal retinas and 37 bundles in 10 treated retinas were examined. In normal retinas, the stain of axonal cytoskeleton was approximately uniform within bundles, and RNFL birefringence did not change along bundles. In treated retinas, elevation of IOP caused non-uniform alteration of axonal cytoskeleton across the retina, and distortion of axonal MTs was associated with decreased birefringence. The study further demonstrated that change of RNFL birefringence profiles along bundles can imply altered axonal cytoskeleton, suggesting that ultrastructural change of the RNFL can be inferred from clinical measurements of RNFL birefringence. The study also demonstrated that measuring RNFL birefringence profiles along bundles, instead of at a single location, may provide a more sensitive way to detect axonal ultrastructural change. CONCLUSIONS: Measurement of RNFL birefringence along bundles can provide estimation of cytoskeleton alteration and sensitive detection of glaucomatous damage.

Reflectance Spectrum and Birefringence of the Retinal Nerve Fiber Layer With Hypertensive Damage of Axonal Cytoskeleton.

Purpose: Glaucoma damages the retinal nerve fiber layer (RNFL). This study used precise multimodal image registration to investigate the changes of the RNFL reflectance spectrum and birefringence in nerve fiber bundles with different degrees of axonal damage. Methods: The reflectance spectrum of individual nerve fiber bundles in normal rats and rats with experimental glaucoma was measured from 400 to 830 nm and their birefringence was measured at 500 nm. Optical measurements of the same bundles were made at different distances from the optic nerve head (ONH). After the optical measurements, the axonal cytoskeleton of the RNFL was evaluated by confocal microscopy to assess the severity of cytoskeletal change. Results: For normal bundles, the shape of the RNFL reflectance spectrum and the value of RNFL birefringence did not change along bundles. In treated retinas, damage to the cytoskeleton varied within and across retinas. The damage in retinal sectors was subjectively graded from normal-looking to severe. Change of spectral shape occurred near the ONH in all sectors studied. This change became more prominent and occurred farther from the ONH with increased damage severity. In contrast, RNFL birefringence did not show change in normal-looking sectors, but decreased in sectors with mild and moderate damage. The birefringence of severely damaged sectors was either within or below the normal range. Conclusions: Varying degrees of cytoskeletal damage affect the RNFL reflectance spectrum and birefringence differently, supporting differences in the ultrastructural basis for the two optical properties. Both properties, however, may provide a means to detect disease and to estimate ultrastructural damage of the RNFL in glaucoma.

Microtubule contribution to the reflectance of the retinal nerve fiber layer.

PURPOSE: The reflectance of the retinal nerve fiber layer (RNFL) arises from light scattering by cylindrical structures oriented parallel to ganglion cell axons. In amphibian retinas, at 440 nm, microtubules (MTs) contribute about one half of RNFL reflectance. In rodent retinas, MTs are the only structure contributing to RNFL birefringence. To increase understanding of the anatomic basis for clinical RNFL measurements, this study was conducted to evaluate the MT contribution to RNFL reflectance in rodent retinas by using the MT depolymerizing agent colchicine. METHODS: Reflectance of nerve fiber bundles in isolated rat retinas was measured at 460, 580, and 830 nm with a multispectral imaging reflectometer. Images were taken frequently over an extended period. During baseline, the tissue was perfused with a physiological solution. During a treatment period, the solution was switched either to a control solution or to a solution containing colchicine. RESULTS: Because of the high reflectance of the RNFL, nerve fiber bundles appeared as bright stripes against a darker retina. The reflectance of bundles was relatively stable in control experiments. With colchicine treatment, however, bundle reflectance at first decreased rapidly and then became stable. After 70 minutes of colchicine treatment, RNFL reflectance had declined to approximately 50% below baseline at all wavelengths. CONCLUSIONS: MTs contribute to RNFL reflectance at all wavelengths. Unlike RNFL birefringence, however, which totally disappears after colchicine treatment, about one half of RNFL reflectance remained after colchicine treatment. This result suggests that, in addition to MTs, other mechanisms may contribute to RNFL reflectance.

Registration of high-density cross sectional images to the fundus image in spectral-domain ophthalmic optical coherence tomography.

We previously developed a technique to acquire a SLO (scanning laser ophthalmoscope) like fundus intensity image from the raw spectra measured with spectral-domain optical coherence tomography (OCT), the same spectra used to generate a 3D OCT data set. This technique offers simultaneous fundus and OCT images and, therefore, solves the problem of registering a cross sectional OCT image to fundus features. However, the registration of high density OCT images is still an unsolved problem because no useful fundus image can be generated from the high density scans. High density OCT images can significantly improve the image quality and enhance the visualization of retinal structure, especially the structure of small lesions. We have developed a feature-based algorithm, which can register a high density OCT image on the fundus image generated from normal density scans. The algorithm was successfully tested for both normal and diseased eyes.

Retinal nerve fiber layer reflectometry must consider directional reflectance.

Recent studies reveal that measurements of retinal nerve fiber layer (RNFL) reflectance provide more sensitive detection of glaucomatous damage than RNFL thickness, but most do not consider directional reflectance of the RNFL, an important source of variability. This study quantitatively compared RNFL directional reflectance, represented by an angular spread function (ASF), measured at different scattering angles, different wavelengths and different distances from the optic nerve head (ONH) and for bundles with different thicknesses (T). An ASF was characterized by its amplitude (A) and width (W). Internal reflectance of a bundle was expressed as A/T. The study found that A varied significantly with scattering angle and wavelength and that A/T was different among bundles but constant along the same bundle, indicating that the internal structure of axons may vary among bundles but does not change with distance. This study also found that W was larger near the ONH and at longer wavelengths, but did not depend on scattering angle or T. Because a 4.3° change in incident angle can change reflected intensity by a factor of 2.7, accounting for directional reflectance should improve the accuracy and reproducibility of RNFL reflectance measurements.

Microtubules contribute to the birefringence of the retinal nerve fiber layer.

PURPOSE: The retinal nerve fiber layer (RNFL) exhibits birefringence that is due to the oriented cylindrical structure of the ganglion cell axons. Possible birefringent structures include axonal membranes, microtubules (MTs), and neurofilaments. MTs are generally assumed to be a major contributor, but this has not been demonstrated. In this study, the MT depolymerizing agent colchicine was used to evaluate the contribution of MTs to RNFL birefringence. METHODS: Retinal nerve fiber bundles of isolated rat retina were observed through an imaging polarimeter set near extinction. Images were taken over an extended period. During baseline, the tissue was perfused with a physiological solution. During a treatment period, the solution was switched either to a control solution identical with the baseline solution or to a similar solution containing colchicine. The contrast of nerve fiber bundles was used to follow change of RNFL birefringence over time. RESULTS: When imaged by the polarimeter, birefringent retinal nerve fiber bundles appeared as either bright or dark stripes. Bundles displayed as bright stripes were used to follow changes in retardance. The contrast of nerve fiber bundles was stable in control experiments. However, in treatment experiments, bundles were bright during the baseline period, but the contrast of bundles decreased rapidly when the colchicine solution was applied; bundles were barely visible after 30 minutes of treatment. After 70 minutes, the bundle contrast was close to zero at all wavelengths studied (440-780 nm). CONCLUSIONS: MTs make a significant contribution to RNFL birefringence. The decrease of RNFL birefringence in glaucoma may indicate a loss of MTs.

Reproducibility of retinal nerve fiber thickness measurements using the stratus OCT in normal and glaucomatous eyes.

PURPOSE: To determine the reproducibility of Stratus Optical Coherence Tomography (OCT) retinal nerve fiber layer (RNFL) measurements around the optic nerve in normal and glaucomatous eyes. METHODS: One eye was chosen at random from 88 normal subjects and 59 glaucomatous subjects distributed among mild, moderate, and severe glaucoma, determined by visual field testing. Subjects underwent six RNFL thickness measurements performed by a single operator over a 30-minute period with a brief rest between sessions. Three scans were taken with the high-density Standard RNFL protocol, and three were taken with the Fast RNFL protocol, alternating between scan protocols. RESULTS: Reliability, as measured by intraclass correlation coefficient (ICC), was calculated for the overall mean RNFL thickness and for each quadrant. The ICC for the mean Standard RNFL thickness (and lower 95% confidence interval [CI]) in normal and glaucomatous eyes was 0.97 (0.96 CI) and 0.98 (0.97 CI), respectively. The ICC for the mean Fast RNFL thickness in normal and glaucomatous eyes was 0.95 (0.93 CI) and 0.97 (0.95 CI), respectively. Quadrant ICCs ranged between 0.79 and 0.97, with the nasal quadrant being the least reproducible of all four quadrants, using either the Standard or Fast RNFL program. The test-retest variability ranged from 3.5 microm for the average RNFL thickness measurements in normal eyes to 13.8 microm for the nasal quadrant measurements in glaucomatous eyes, which appeared to be the most variable. CONCLUSIONS: Reproducibility of RNFL measurements using the Stratus OCT is excellent in normal and glaucomatous eyes. The nasal quadrant appears to be the most variable measurement. Standard RNFL and Fast RNFL scans are equally reproducible and yield comparable measurements. These findings have implications for the diagnosis of glaucoma and glaucomatous progression.