Panretinal photocoagulation is used to treat diabetic retinopathy. Measurements with SLO and microspheres show that the choriocapillaris is locally damaged under lesions in the cat retina. This damage could limit the effectiveness of photocoagulation.
To investigate the effects of argon laser photocoagulation on the choroidal circulation in cats.
Three sizes of argon laser lesions designed to damage the outer retina were created in six cats: larger than 1 mm, 500 μm, and 200 μm. At least 1 month after the lesions, damage to the choroidal vasculature was studied in two ways. First, scanning laser ophthalmoscopy was used to obtain infrared reflectance (IR) photographs and indocyanine green (ICG) angiograms. Second, fluorescent microspheres (15 μm) were injected into the left ventricle. The globes were fixed, the choroid was flat mounted, and images were taken with a fluorescence microscope. Retinal histology was assessed in comparable lesions.
Histology showed that the inner retina was preserved, but the choroid, tapetum, and outer retina were damaged. ICG angiograms revealed choriocapillaris loss in large lesions and in some 500-μm lesions, whereas the larger vessels were preserved; in 200 μm lesions, choriocapillaris loss was not detectable. However, in all lesions, the distribution of microspheres revealed little if any choriocapillaris flow. In larger lesions, the damaged region was surrounded by an area in which the number of microspheres was higher than in the lesion but lower than in the normal retina.
Under lesions that destroyed photoreceptors, the choriocapillaris was also compromised, even when no change could be detected with ICG angiography. Panretinal photocoagulation is designed to increase retinal Po2 by allowing choroidal oxygen to reach the inner retina, but its effectiveness may be limited by damage to the choriocapillaris.
Background: With the advent of confocal scanning laser ophthalmoscopes (cSLO), fundus autofluorescence (FAF) resulting mainly from lipofuscin accumulation on the level of the retinal pigment epithelium can be visualised in vivo. Various cSLOs are available to document FAF. The authors analysed and compared results of FAF using three different instruments.
Methods: Eight eyes of eight normal volunteers and 18 eyes of 12 patients with different retinal diseases (age related macular degeneration, macular dystrophy, central serous retinopathy) were examined. FAF images were recorded from each subject with the Heidelberg retina angiograph (HRA), the Rodenstock cSLO (RcSLO) and the Zeiss Prototype SM 30-4024 (ZcSLO). For excitation an argon laser (488 nm) was used (barrier filter: HRA 500 nm; RcSLO 515 nm; ZcSLO 521 nm). 32 FAF images were aligned and averaged using the same software for all cSLOs. FAF distribution was measured and grey scale values as well as root mean square (RMS) contrast were compared.
Results: Mean age of all subjects was 55.5 (SD 21.4) years. The maximum grey scale value averaged across all eyes was 76.19 (39.34) for the HRA, 61.44 (22.12) for the ZcSLO and 37.0 (9.97) for the RcSLO. The RMS contrast was 0.46 (0.20) for the ZcSLO, 0.40 (0.12) for the HRA, and 0.13 (0.05) for the RcSLO. The differences between the cSLOs were statistically significant with higher grey scale levels and more contrast for the HRA and ZcSLO than the RcSLO (repeated measures ANOVA; p<0.0001). The differences between the HRA and the ZcSLO were not significant (post hoc comparisons; p<0.05).
Conclusions: All cSLOs allow clinically useful FAF imaging in retinal diseases. However, grey scale levels and contrast were much lower on the RcSLO. Therefore, RcSLO images appear much darker than HRA or ZcSLO images. Furthermore, not all cSLOs have a fixed photodetector gain and a standardised value for the argon laser amplification, which is mandatory for an absolute comparison of FAF imaging results.
scanning laser ophthalmoscope; fundus autofluorescence; lipofuscin; retinal pigment epithelium; macular degeneration
Background: With the advent of digital confocal scanning laser ophthalmoscopy it is possible to detect low levels of fluorescence. Here we used a novel confocal scanning laser ophthalmoscope (cSLO) to determine lower limits of dye required for fluorescein (FL) and indocyanine green (ICG) angiography.
Methods: A cSLO (Heidelberg retina angiograph 2, Heidelberg Engineering, Dossenheim, Germany) with an optically pumped solid state laser (488 nm) for FL and a diode laser (790 nm) for ICG angiography (FL/ICG-A) was used. 62 FL-As were performed in 53 patients and 45 ICG-As were performed in 39 patients with neovascular age related macular degeneration. The volume and overall dye content of bolus injections was gradually tapered (FL: 500 mg, 250 mg, 200 mg, 166 mg, 100 mg; ICG: 25 mg, 20 mg, 15 mg, 10 mg, 5 mg, 2.5 mg), while dye concentrations were kept constant at 100 mg/ml for FL and at 5 mg/ml for ICG. Images were obtained 1, 5, 15, and 30 minutes after dye injection. Image quality was evaluated by two independent readers using standardised criteria.
Results: For amounts down to 166 mg for FL and to 5 mg for ICG, sufficient image quality was achieved during all phases following injection. Only late phase images showed less contrast compared to typically used dye amounts, which was irrelevant for interpretation and clinical management.
Conclusions: With the increased sensitivity of this novel cSLO system, amounts of injected dye during FL-A can be reduced to one third for FL and to one fifth for ICG without relevant loss of image quality or information compared to conventionally used dye levels. These amounts can be used for routine angiography and allow relevant savings for units performing FL-A.
confocal scanning laser ophthalmoscopy; fluorescence angiography; fluorescein; indocyanine green
An infrared scanning laser ophthalmoscope (SLO) has been custom built in order to investigate the application of confocal and indirect mode SLO imaging to patients with fundus disease. Infrared light is reflected from the fundus to a greater extent than visible light permitting lower illumination power and, as it penetrates the retinal pigment epithelium, choroidal structures can be readily imaged. Furthermore, as conventional infrared illumination and detection systems are not well suited to ophthalmoscopy, this area is underdeveloped as a potential source of useful clinical data. Confocal, direct and indirect imaging modes have been used to image fundi of normal volunteers and patients with fundus disease. In comparison with conventional fundus photography confocal infrared SLO imaging improves visualisation of choroidal vasculature, retinal pigment epithelial abnormalities, laser photocoagulation scars, and optic disc pores in the lamina cribrosa. Direct infrared SLO imaging enables fundus visualisation through nuclear lens opacities. Furthermore, indirect mode imaging enhances significantly the appearance of macular drusen. The potential clinical benefit of these observations is discussed.
BACKGROUND—Optic nerve head drusen may present diagnostic difficulties in cases of disc swelling. Imaging of the nerve in a search for drusen is often inconclusive, especially in children, where drusen may be buried below the surface of the nerve head.
METHODS—A small study was carried out using a scanning laser ophthalmoscope (SLO) with an infrared confocal facility to scan deep within optic discs in an attempt to image drusen.
RESULTS—The SLO was able to demonstrate superficial and buried drusen (using the infrared confocal facility). The superiority of the SLO over ultrasound in the presence of lens opacity was revealed, as the SLO simultaneously demonstrated both drusen and the associated anomalous disc features which are not detected by ultrasound.
CONCLUSION—The SLO can help in the diagnosis of optic disc drusen especially in difficult cases where lens opacity or buried drusen hinders their definitive diagnosis.
To conduct high-resolution imaging of the retinal nerve fiber layer (RNFL) in normal eyes using adaptive optics scanning laser ophthalmoscopy (AO-SLO).
AO-SLO images were obtained in 20 normal eyes at multiple locations in the posterior polar area and a circular path with a 3–4-mm diameter around the optic disc. For each eye, images focused on the RNFL were recorded and a montage of AO-SLO images was created.
AO-SLO images for all eyes showed many hyperreflective bundles in the RNFL. Hyperreflective bundles above or below the fovea were seen in an arch from the temporal periphery on either side of a horizontal dividing line to the optic disc. The dark lines among the hyperreflective bundles were narrower around the optic disc compared with those in the temporal raphe. The hyperreflective bundles corresponded with the direction of the striations on SLO red-free images. The resolution and contrast of the bundles were much higher in AO-SLO images than in red-free fundus photography or SLO red-free images. The mean hyperreflective bundle width around the optic disc had a double-humped shape; the bundles at the temporal and nasal sides of the optic disc were narrower than those above and below the optic disc (P<0.001). RNFL thickness obtained by optical coherence tomography correlated with the hyperreflective bundle widths on AO-SLO (P<0.001)
AO-SLO revealed hyperreflective bundles and dark lines in the RNFL, believed to be retinal nerve fiber bundles and Müller cell septa. The widths of the nerve fiber bundles appear to be proportional to the RNFL thickness at equivalent distances from the optic disc.
We have recently described a novel way of imaging apoptosing retinal ganglion cells in vivo in the rat. This study investigated if this technique could be used in the mouse, and whether the Heidelberg Retina Angiograph II (HRAII) was appropriate.
Retinal ganglion cell (RGC) death was induced by intravitreal injections in rat and mouse eyes using staurosporine. Fluorescent-labeled apoptosing cells were detected by imaging with both the HRAII and a prototype Zeiss confocal scanning laser ophthalmoscope (cSLO). Averaged in vivo images were analyzed and results compared with histologic analysis.
Fluorescent points (FPs) used as a measure of RGC apoptosis in vivo were detected in the mouse eye but only with the HRAII and not the Zeiss cSLO. The HRAII was able to detect 62% more FPs in rat than the Zeiss cSLO. Both cSLOs showed peak FP counts at the 5- to 10-μm range in rat and mouse. Maximal FP counts were detected in the superior and superior temporal regions in the rat, with no obvious pattern of distribution in the mouse. The HRAII was found to have more FP correspondence with histologically identified apoptosing RGCs.
To our knowledge, this is the first demonstration of visualized apoptosing RGC in vivo in a mouse. The improved image quality achieved with the HRAII compared with the Zeiss cSLO was validated by histology. This together with its enhanced maneuverability and the fact that it is already commercially available make the HRAII a potential tool for the early detection and diagnosis of glaucomatous disease in patients.
apoptosis; in vivo imaging; mouse model; retinal ganglion cell
Aim: To investigate en face optical coherence tomography (eOCT) and its use as an effective objective technique for assessing changes in the glaucomatous rat optic nerve head (ONH) in vivo, and compare it with confocal scanning laser ophthalmoscopy (cSLO).
Methods: 18 Dark Agouti (DA) rats with surgically induced ocular hypertension were imaged with eOCT and cSLO at regular intervals. Assessment included three dimensional (3D) topographic reconstructions, intensity z-profile plots, a new method of depth analysis to define a “multilayered” structure, and scleral canal measurements, in relation to the degree of intraocular pressure (IOP) exposure.
Results: The increased depth resolution of the eOCT compared to the cSLO was apparent in all methods of analysis, with better discrimination of tissue planes. This was validated histologically. eOCT demonstrated several significant changes in imaged rat ONH which correlated with IOP exposure, including the area of ONH (p<0.01), separation between retinal vessel and scleral layers (p<0.05), and anterior scleral canal opening expansion (p<0.05).
Conclusion: eOCT appears to be effective in assessing rat ONH, allowing detailed structural analysis of the multilayered ONH structure. As far as the authors are aware, this is the first report of scleral canal expansion in a rat model. They suggest eOCT as a novel method for the detection of early changes in the ONH in glaucoma.
image analysis; optic disc; glaucoma; intraocular pressure; animal experimentation
Glaucoma damages 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.
Retinal Nerve Fiber Layer; Animal Model of Glaucoma; Cytoskeleton; F-actin; Phalloidin Staining; Confocal Laser Scanning Microscopy
A method of quantifying fundus autofluorescence with a scanning laser ophthalmoscope was developed and validated. It allows measurement of disease progression, effects of treatment, and age-related changes.
To evaluate the feasibility and reliability of a standardized approach for quantitative measurements of fundus autofluorescence (AF) in images obtained with a confocal scanning laser ophthalmoscope (cSLO).
AF images (30°) were acquired in 34 normal subjects (age range, 20–55 years) with two different cSLOs (488-nm excitation) equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The gray levels (GLs) of each image were calibrated to the reference, the zero GL, and the magnification, to give quantified autofluorescence (qAF). Images from subjects and fixed patterns were used to test detector linearity with respect to fluorescence intensity, the stability of qAF with change in detector gain, field uniformity, effect of refractive error, and repeatability.
qAF was independent of detector gain and laser power over clinically relevant ranges, provided that detector gain was adjusted to maintain exposures within the linear detection range (GL < 175). Field uniformity was better than 5% in a central 20°-diameter circle but decreased more peripherally. The theoretical inverse square magnification correction was experimentally verified. Photoreceptor bleaching for at least 20 seconds was performed. Repeatability (95% confidence interval) for same day and different-day retests of qAF was ±6% to ±14%. Agreement (95% confidence interval) between the two instruments was <11%.
Quantitative AF imaging appears feasible. It may enhance understanding of retinal degeneration, serve as a diagnostic aid and as a sensitive marker of disease progression, and provide a tool to monitor the effects of therapeutic interventions.
The impact of retinal pathology detected by high-resolution imaging on vision remains largely unexplored. Therefore, the aim of the study was to achieve high-resolution structure-function correlation of the human macula in vivo.
To obtain high-resolution tomographic and topographic images of the macula spectral-domain optical coherence tomography (SD-OCT) and confocal scanning laser ophthalmoscopy (cSLO), respectively, were used. Functional mapping of the macula was obtained by using fundus-controlled microperimetry. Custom software allowed for co-registration of the fundus mapped microperimetry coordinates with both SD-OCT and cSLO datasets. The method was applied in a cross-sectional observational study of retinal diseases and in a clinical trial investigating the effectiveness of intravitreal ranibizumab in macular telangietasia type 2. There was a significant relationship between outer retinal thickness and retinal sensitivity (p<0.001) and neurodegeneration leaving less than about 50 µm of parafoveal outer retinal thickness completely abolished light sensitivity. In contrast, functional preservation was found if neurodegeneration spared the photoreceptors, but caused quite extensive disruption of the inner retina. Longitudinal data revealed that small lesions affecting the photoreceptor layer typically precede functional detection but later cause severe loss of light sensitivity. Ranibizumab was shown to be ineffective to prevent such functional loss in macular telangietasia type 2.
Since there is a general need for efficient monitoring of the effectiveness of therapy in neurodegenerative diseases of the retina and since SD-OCT imaging is becoming more widely available, surrogate endpoints derived from such structure-function correlation may become highly relevant in future clinical trials.
AIMS—To determine the sensitivity of confocal scanning laser ophthalmoscopy (SLO) in detecting clinically significant changes in papilloedema secondary to idiopathic intracranial hypertension (IIH) and the correlation with visual field loss.
METHODS—Eight patients—three new, two recurrent, and three chronic cases of IIH—were examined over a 9 month period with SLO (Heidelberg retina tomograph) of optic nerve head and 30-2 Humphrey visual fields (six cases). Optic disc swelling (volume) was assessed in each eye using a circular contour line placed around the swollen optic nerve head on the mean image of three topographic images. Nine volume measurements from single images in each eye of every patient were performed on one occasion to assess repeatability.
RESULTS—In the five acute cases optic disc volumes (range 1-16 mm3) decreased with treatment to stable, normal levels. Three of these had mild, reproducible, field defects which resolved. Two chronic cases had stable or fluctuating disc volume with no detectable change in grade of papilloedema and mild field loss. In one case which underwent theco-peritoneal shunting both disc volume and field worsened, indicating therapeutic failure. Both improved postoperatively.
CONCLUSIONS—SLO has a high sensitivity for detecting small changes in disc volumes and correlates closely with visual field change in the short term. It can confirm therapeutic failure by detecting stable or increasing disc volume. Decreasing volume may indicate resolution of papilloedema or secondary optic atrophy, so accompanying funduscopy and visual fields remain essential.
Keywords: intracranial hypertension; scanning laser ophthalmoscopy; visual field; optic disc
Scanning laser ophthalmoscopy (SLO) and spectral domain optical coherence tomography (SDOCT) have become essential clinical diagnostic tools in ophthalmology by allowing for video-rate noninvasive en face and depth-resolved visualization of retinal structure. Current generation multimodal imaging systems that combine both SLO and OCT as a means of image tracking remain complex in their hardware implementations. Here, we combine a spectrally encoded confocal scanning laser ophthalmoscope (SECSLO) with an ophthalmic SDOCT system. This novel implementation of an interlaced SECSLO-SDOCT system allows for video-rate SLO fundus images to be acquired alternately with high-resolution SDOCT B-scans as a means of image aiming, guidance, and registration as well as motion tracking. The system shares the illumination source, detection system, and scanning optics between both SLO and OCT as a method of providing a simple multimodal ophthalmic imaging system that can readily be implemented as a table-top or hand-held device.
(110.4234) Multispectral and hyperspectral imaging; (170.1790) Confocal microscopy; (170.3880) Medical and biological imaging; (170.4460) Ophthalmic optics and devices; (170.4500) Optical coherence tomography
Our study was conducted to establish procedures and protocols for quantitative autofluorescence (qAF) measurements in mice, and to report changes in qAF, A2E bisretinoid concentration, and outer nuclear layer (ONL) thickness in mice of different genotypes and age.
Fundus autofluorescence (AF) images (55° lens, 488 nm excitation) were acquired in albino Abca4−/−, Abca4+/−, and Abca4+/+ mice (ages 2–12 months) with a confocal scanning laser ophthalmoscope (cSLO). Gray levels (GLs) in each image were calibrated to an internal fluorescence reference. The bisretinoid A2E was measured by quantitative high performance liquid chromatography (HPLC). Histometric analysis of ONL thicknesses was performed.
The Bland-Altman coefficient of repeatability (95% confidence interval) was ±18% for between-session qAF measurements. Mean qAF values increased with age (2–12 months) in all groups of mice. qAF was approximately 2-fold higher in Abca4−/− mice than in Abca4+/+ mice and approximately 20% higher in heterozygous mice. HPLC measurements of the lipofuscin fluorophore A2E also revealed age-associated increases, and the fold difference between Abca4−/− and wild-type mice was more pronounced (approximately 3–4-fold) than measurable by qAF. Moreover, A2E levels declined after 8 months of age, a change not observed with qAF. The decline in A2E levels in the Abca4−/− mice corresponded to reduced photoreceptor cell viability as reflected in ONL thinning beginning at 8 months of age.
The qAF method enables measurement of in vivo lipofuscin and the detection of genotype and age-associated differences. The use of this approach has the potential to aid in understanding retinal disease processes and will facilitate preclinical studies.
We established a standardized approach for quantitative autofluorescence (qAF) measurements in mice, and report changes in qAF, A2E bisretinoid concentration, and outer nuclear layer (ONL) thickness in mice of different genotypes and age.
Abca4; RPE lipofuscin; quantitative fundus autofluorescence; mouse; bisretinoid
To provide an update on the role of optic nerve and peripapillary retinal nerve fiber layer imaging in glaucoma clinical practice and clinical trials.
Review of recent literature and authors’ clinical and laboratory studies.
Imaging technologies such as confocal scanning laser ophthalmoscopy, scanning laser polarimetry, and optical coherence tomography provide objective and quantitative measurements that are highly reproducible and show very good agreement with clinical estimates of optic nerve head structure and visual function. Structural assessments provided by imaging complement optic disc photography in clinical care, and have the potential to identify relevant structural efficacy endpoints in glaucoma randomized clinical trials. As with other technologies, imaging may produce false identification of glaucoma and its progression, thus clinicians should not make management decisions based solely on the results of one single test or technology.
Although optic disc stereophotography represents the standard for documentation of glaucomatous structural damage in practice and research trials, advances in computerized imaging technology provide useful measures that assist the clinician in glaucoma diagnosis and monitoring, and offer considerable opportunity for use as efficacy endpoints in clinical trials.
Retinal vascular diseases are a leading cause of blindness and visual disability.
The advent of adaptive optics retinal imaging has enabled us to image the
retinal vascular at cellular resolutions, but imaging of the vasculature can be
difficult due to the complex nature of the images, including features of many
other retinal structures, such as the nerve fiber layer, glial and other cells.
In this paper we show that varying the size and centration of the confocal
aperture of an adaptive optics scanning laser ophthalmoscope (AOSLO) can
increase sensitivity to multiply scattered light, especially light forward
scattered from the vasculature and erythrocytes. The resulting technique was
tested by imaging regions with different retinal tissue reflectivities as well
as within the optic nerve head.
(110.1085) Adaptive imaging; (110.1220) Apertures; (170.4460) Ophthalmic optics and devices; (170.1470) Blood or tissue constituent monitoring
To define differences in optic disc, retinal nerve fiber layer, and macular structure between healthy participants of African (AD) and European descent (ED) using quantitative imaging techniques in the African Descent and Glaucoma Evaluation Study (ADAGES).
Reliable images were obtained using stereoscopic photography, confocal scanning laser ophthalmoscopy (Heidelberg retina tomography [HRT]), and optical coherence tomography (OCT) for 648 healthy subjects in ADAGES. Findings were compared and adjusted for age, optic disc area, and reference plane height where appropriate.
The AD participants had significantly greater optic disc area on HRT (2.06 mm2; P<.001) and OCT (2.47 mm2; P<.001) and a deeper HRT cup depth than the ED group (P<.001). Retinal nerve fiber layer thickness was greater in the AD group except within the temporal region, where it was significantly thinner. Central macular thickness and volume were less in the AD group.
Most of the variations in optic nerve morphologic characteristics between the AD and ED groups are due to differences in disc area. However, differences remain in HRT cup depth, OCT macular thickness and volume, and OCT retinal nerve fiber layer thickness independent of these variables. These differences should be considered in the determination of disease status.
AIMS--An investigation was carried out to compare the image quality of the ocular fundus obtained clinically, photographically, and with the scanning laser ophthalmoscope (SLO) at visible and infrared wavelengths in patients with significant cataract. METHODS--Nineteen patients admitted for routine cataract extraction were examined clinically by two independent observers to ascertain cataract type and clarity of fundus view with an indirect ophthalmoscope. Fundus photography and both confocal and direct (non-confocal) SLO imaging at 590 nm, 670 nm, and 830 nm were carried out after pupillary dilatation. Images obtained were graded independently using a recognised grading system. RESULTS--Quality of SLO images appeared to be superior to indirect ophthalmoscopy (p < 0.01) and fundus photography (p < 0.001) when graded subjectively. Quantitative analysis of contrast of retinal vessels demonstrated significantly higher contrast for the SLO compared with digitised fundus photographs at all wavelengths tested (p < 0.001), with highest contrast at 590 nm. Use of a confocal aperture significantly improved vessel contrast but may reduce overall image intensity. CONCLUSIONS--Scanning laser ophthalmoscopy may offer a method to observe and record fine fundus detail in patients who have marked cataract.
Anterior lamina cribrosa pores were consistently imaged in vivo in normal human and macaque eyes across imaging sessions using adaptive optics (AO). Results suggest that AO imaging can be used to track laminar pore changes in vivo in glaucoma.
The ability to consistently resolve lamina cribrosa pores in vivo has applications in the study of optic nerve head and retinal disease mechanisms. Repeatability was assessed in imaging laminar pores in normal living eyes with a confocal adaptive optics scanning laser ophthalmoscope (AOSLO).
Reflectance images (840 nm) of the anterior lamina cribrosa were acquired using the AOSLO in four or more different sessions in two normal rhesus monkey eyes and three normal human eyes. Laminar pore areas, elongations (ratio of major to minor axes of the best-fit ellipse) and nearest neighbor distances were calculated for each session. Measurement repeatability was assessed across sessions.
Pore areas ranged from 90 to 4365 μm2 in monkeys and 154 to 6637 μm2 in humans. Mean variabilities in measuring pore area and elongation (i.e., mean of the standard deviation of measurements made across sessions for the same pores) were 50 μm2 (6.1%) and 0.13 (6.7%), respectively, in monkeys and 113 μm2 (8.3%) and 0.17 (7.7%), respectively, in humans. Mean variabilities in measuring nearest neighbor distances were 1.93 μm (5.2%) in monkeys and 2.79 μm (4.1%) in humans. There were no statistically significant differences in any pore parameters across sessions (ANOVA, P > 0.05).
The anterior lamina cribrosa was consistently imaged in vivo in normal monkey and human eyes. The small intersession variability in normal pore geometry suggests that AOSLO imaging could be used to measure and track changes in laminar pores in vivo during glaucomatous progression.
This article describes the distribution of major cytoskeleton components in the retinal nerve fiber layer (RNFL) during the development of glaucoma. The study found that distortion of axonal cytoskeleton can occur before the thinning of the RNFL.
Glaucoma damages the retinal nerve fiber layer (RNFL). The purpose of this study was to investigate the distribution of major cytoskeleton components, F-actin, microtubules (MTs), and neurofilaments (NFs), in the RNFL during the development of glaucoma.
Intraocular hypertension was induced in a rat model by laser photocoagulation of the trabecular meshwork. Retinas were obtained after 2 to 3.5 weeks of treatment. Multiple fluorescent stains were used to identify F-actin, MTs, NFs, and nuclei simultaneously in the same tissue. Distribution of these components in a whole-mounted retina was examined by confocal microscopy. Fluorescent stain was quantitatively described.
In normal RNFL F-actin, MTs, and NFs were intensely stained. Along the bundles, F-actin and MTs were strongly colocalized, but alternating strands of F-actin and NFs were apparent. Normal RNFL lacked nuclei. In glaucomatous retinas, irregular staining of F-actin, MTs, and NFs was found within the bundles. A strong network of F-actin appeared on the RNFL surface and between the bundles. In severely damaged retinal regions total loss of F-actin and MTs was found, whereas residual strands of NFs were evident. Before the decrease in RNFL thickness, irregularity of F-actin stain and density of nuclei in the RNFL significantly increased.
The results suggest that F-actin, MTs, and NFs are rich and approximately uniformly distributed in the normal RNFL. Glaucoma causes alteration of the cytoskeleton in the RNFL. F-actin is the most sensitive component in its response to stress on the retina. An increase in the number of nuclei in the RNFL may be an early sign of glaucomatous damage
In this study, a decrease in RNFL reflectance was found near the ONH in glaucomatous eyes. The change preceded thinning of the RNFL, suggesting that a decrease in RNFL reflectance near the ONH is an early sign of glaucomatous damage.
Glaucoma damages the retinal never fiber layer (RNFL). RNFL thickness, measured with optical coherence tomography (OCT), is often used in clinical assessment of the damage. In this study the relation between the RNFL reflectance and thickness at early stages of glaucoma was investigated.
A rat model of glaucoma was used that involved laser photocoagulation of the trabecular meshwork. The reflectance of the RNFL in an isolated retina was measured, followed by immunohistochemical staining of the axonal cytoskeleton. RNFL thickness was measured by confocal fluorescence imaging. RNFL reflectance was calculated for bundle areas located at radii of 0.22, 0.33, and 0.44 mm from the optic nerve head (ONH) center. Linear regression was used to study the relation between reflectance and thickness. For glaucomatous eyes, only those bundles with no apparent structural damage were used.
Bundles in 11 control retinas and 10 treated retinas were examined. Bundle thickness of both groups at each radius was similar (P = 0.89). The reflectance of the bundles at radii of 0.33 and 0.44 mm was found to be similar in both control and treated retinas (P > 0.5). However, the reflectance of the bundles at the 0.22-mm radius decreased significantly in the treated group (P = 0.005).
Elevation of intraocular pressure causes decrease in RNFL reflectance for bundles near the ONH. Change in RNFL reflectance precedes thinning of the RNFL. The results suggest that a decrease in RNFL reflectance near the ONH is an early sign of glaucomatous damage.
AIMS—Conventional fundus imaging using a fundus camera produces colour fundus pictures. The scanning laser ophthalmoscope (SLO) has the advantages of lower levels of light exposure, improved contrast, and direct digital imaging but until now has produced monochromatic images as a laser of single wavelength is used. True representation of the fundus is possible by combining images taken using blue, green, and red lasers.
METHODS—A custom built SLO was used to capture blue, green, and red fundus images from suitable volunteers and patients with fundus disease. Images were corrected for eye movement and combined to form a colour image. Colour fundus photographs were taken using a fundus camera for comparison with the SLO image.
RESULTS—The background fundus and retinal vasculature had similar appearances with the two imaging modalities. Internal limiting membrane reflections were prominent with the SLO. Identification of new vessels in the diabetic fundus was easier with the SLO than the colour fundus photographs.
CONCLUSION—A colour SLO offers all the advantages of the present monochromatic imaging system with the added advantage of true colour representation of the fundus.
Keywords: scanning laser ophthalmoscope; fundus imaging; digital colour fundus images
In vivo two-photon imaging through the pupil of the primate eye has the potential to become a useful tool for functional imaging of the retina. Two-photon excited fluorescence images of the macaque cone mosaic were obtained using a fluorescence adaptive optics scanning laser ophthalmoscope, overcoming the challenges of a low numerical aperture, imperfect optics of the eye, high required light levels, and eye motion. Although the specific fluorophores are as yet unknown, strong in vivo intrinsic fluorescence allowed images of the cone mosaic. Imaging intact ex vivo retina revealed that the strongest two-photon excited fluorescence signal comes from the cone inner segments. The fluorescence response increased following light stimulation, which could provide a functional measure of the effects of light on photoreceptors.
(010.1080) adaptive optics; (330.4460) Ophthalmic optics and devices; (330.5310) Vision – photoreceptors; (330.7327) Visual optics, ophthalmic instrumentation
To compare optic disc and retinal nerve fiber layer (RNFL) imaging methods to discriminate eyes with early glaucoma from normal eyes.
Retrospective, cross-sectional study.
Setting: Tertiary care academic glaucoma center. Ninety-two eyes of 92 subjects (46 with early perimetric open-angle glaucoma and 46 controls) were studied. Diagnostic performance of optical coherence tomography (StratusOCT), scanning laser polarimetry (GDx-VCC), confocal laser ophthalmoscopy (Heidelberg Retinal Tomograph III), and qualitative assessment of stereoscopic optic disc photographs were compared. Outcome measures were areas under receiver operator characteristic curves (AUC) and sensitivities at fixed specificities. Classification and Regression Trees (CART) analysis was used to evaluate combinations of quantitative parameters.
The average (±SD) visual field mean deviation for glaucomatous eyes was −4.0±2.5 dB. Parameters with largest AUCs (±SE) were: average RNFL thickness for StratusOCT (0.96±0.02), nerve fiber indicator for GDx-VCC (0.92±0.03), FSM discriminant function for HRT III (0.91±0.03), and 0.97±0.02 for disc photograph evaluation. At 95% specificity, sensitivity of disc photograph evaluation (90%) was greater than GDx-VCC (p=0.05) and HRT III (p=0.002), but not significantly different than that of StratusOCT (p>0.05). Combination of StratusOCT average RNFL thickness and HRT III cup/disc area with CART produced a sensitivity of 91% and specificity of 96%
StratusOCT, GDx-VCC, and HRT III performed as well as, but not better than, qualitative evaluation of optic disc stereophotographs for detection of early perimetric glaucoma. The combination of StratusOCT average RNFL thickness and HRT III cup/disc area ratio provided a high diagnostic precision.
The purpose of the review is to provide an update on the role of imaging devices in the diagnosis and follow-up of glaucoma with an emphasis on techniques for detecting glaucomatous progression and the newer spectral domain optical coherence tomography instruments. Imaging instruments provide objective quantitative measures of the optic disc and the retinal nerve fiber layer and are increasingly utilized in clinical practice. This review will summarize the recent enhancements in confocal scanning laser ophthalmoscopy, scanning laser polarimetry, and optical coherence tomography with an emphasis on how to utilize these techniques to manage glaucoma patients and highlight the strengths and limitations of each technology. In addition, this review will briefly describe the sophisticated data analysis strategies that are now available to detect glaucomatous change overtime.
Confocal scanning laser ophthalmoscopy; glaucomatous progression; optical coherence tomography; retinal nerve fiber layer; scanning laser polarimetry