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Purpose: This study aims to investigate the potential in vivo relationship between macular pigment (MP) and retinal layers thickness in healthy subjects and dry, non-advanced age-related macular degeneration (AMD)., Methods: An observational, cross-sectional study was conducted. Healthy subjects >40 years and patients with early or intermediate AMD were recruited. Structural OCT and macular pigment optical volume (MPOV) were collected for each subject. Retinal layers parameters were calculated based on the standard early treatment diabetic retinopathy study (ETDRS) map. Additionally, MPOV within 1°, 2°, and 9° of eccentricity was assessed and associated with retinal layers thickness and volume. Linear mixed-effects models were used to test the relationship between MP and structural OCT parameters, while adjusting for known possible confounding factors., Results: A total of 144 eyes of 91 subjects (60.4% females) were evaluated, comprising 43% normal eyes and 57% with early/intermediate AMD. Among the retinal layers, only the outer nuclear layer (ONL) thickness and volume appeared to be associated to higher MP levels. Specifically, the central ONL thickness was identified as a significant predictor of the MPOV 1°(P = 0.04), while the parafoveal ONL thickness (inner ETDRS subfield) was identified as a significant fixed effect on the MPOV 9° (P = 0.037). Age and the presence of drusen or subretinal drusenoid deposits were also tested without showing significant correlations., Conclusions: Among the retinal layers examined, only the ONL thickness demonstrated a significant association with MPOV. Consequently, ONL thickness might serve as a potential biomarker related to MP levels.

Purpose: To assess retinal vein and artery diameters during active and inactive intraocular inflammation in eyes with uveitis., Methods: Color fundus photographs and clinical data of eyes with uveitis collected during two visits (active disease [i.e., T0] and inactive stage [i.e., T1]) were reviewed. The images were semi-automatically analyzed to obtain the central retina vein equivalent (CRVE) and central retina artery equivalent (CRAE). Changes of CRVE and CRAE from T0 to T1 were calculated, and their possible correlation with clinical data, including age, gender, ethnicity, uveitis etiology, and visual acuity, were investigated., Results: Eighty-nine eyes were enrolled in the study. Both CRVE and CRAE reduced from T0 to T1 (P < 0.0001 and P = 0.01, respectively), with active inflammation being able to influence the CRVE and CRAE (P < 0.0001 and P = 0.0004, respectively) after accounting for all other variables. The degree of venular (∆V) and arteriolar (∆A) dilation was influenced only by time (P = 0.03 and P = 0.04, respectively). Best-corrected visual acuity was influenced by time and ethnicity (P = 0.003 and P = 0.0006)., Conclusions: CRVE and CRAE are increased in eyes with active intraocular inflammation regardless of the type of uveitis, and they decrease when the inflammation wears off.

Purpose: To investigate the course of inherited retinal degenerations (IRD) due to mutations in the RPE65 gene., Methods: This longitudinal multicentric retrospective chart-review study was designed to collect best corrected visual acuity (BCVA), Goldman visual field, optical coherence tomography (OCT), and electroretinography (ERG) measurements. The data, including imaging, were collected using an electronic clinical research form and were reviewed at a single center to improve consistency., Results: From an overall cohort of 60 Italian patients with RPE65-associated IRD, 43 patients (mean age, 27.8 ± 19.7 years) were included and showed a mean BCVA of 2.0 ± 1.0 logMAR. Time-to-event analysis revealed a median age of 33.8 years and 41.4 years to reach low vision and blindness based on BCVA, respectively. ERG (available for 34 patients) showed undetectable responses in most patients (26; 76.5%). OCT (available for 31 patients) revealed epiretinal membranes in five patients (16.1%). Central foveal thickness significantly decreased with age at a mean annual rate of -0.6%/y (P = 0.044). We identified 43 different variants in the RPE65 gene in the entire cohort. Nine variants were novel. Finally, to assess genotype-phenotype correlations, patients were stratified according to the number of RPE65 loss-of-function (LoF) alleles. Patients without LoF variants showed significantly (P < 0.05) better BCVA compared to patients with one or two LoF alleles., Conclusions: We described the natural course of RPE65-associated IRD in an Italian cohort showing for the first time a specific genotype-phenotype association. Our findings can contribute to a better management of RPE65-associated IRD patients.

Purpose: To correlate changes in choroidal thickness and vascularity index with disease activity in patients with neovascular age-related macular degeneration (nAMD)., Methods: Eyes diagnosed with AMD that had two sequential visits within 12 months and that had no choroidal neovascularization (CNV) or had inactive CNV at the first visit were included. Those that had active CNV at follow-up were enrolled as cases. Eyes that did not developed a CNV or that were still inactive at the second visit were enrolled as controls. Disease activity was based on optical coherence tomography (OCT) and fluorescein angiography findings. Subfoveal choroidal thickness (SCT), mean choroidal thickness (MCT), and choroidal vascularity index (CVI) were assessed on enhanced depth imaging OCT and compared between the baseline and follow-up visit. Subgroup analysis accounting for lesion type and previous treatment, if any, were performed., Results: Sixty-five eyes from 60 patients (35 females) and 50 age- and sex-matched controls were included. At the active visit, cases had an increase from 164 ± 67 μm to 175 ± 70 μm in mean ± SD SCT and from 144 ± 45 μm to 152 ± 45 μm in MCT (both P < 0.0001). The mean CVI also increased at from 54.5% ± 3.3% to 55.4% ± 3.8% (P = 0.04). Controls did not show significant changes in choroidal measurements between the two visits. Mean SCT, MCT, and CVI values were similar for previously treated and treatment-naive eyes., Conclusions: Choroidal thickness and CVI significantly increased with active disease in nAMD eyes. Changes in choroidal thickness may predict CNV development or recurrence before they are otherwise evident clinically.

Purpose: The purpose of this study was to evaluate the relationship between blue-fundus autofluorescence (B-FAF) and optical coherence tomography (OCT) in eyes with lamellar macular holes (LMHs)., Methods: this was a multicenter, observational case series. Ninety-two eyes with LMH associated with the standard epiretinal membrane (ERM) or lamellar hole-associated epiretinal proliferation (LHEP) were evaluated. The eyes must also present an area of increased autofluorescence on B-FAF., Results: The ERM-alone group and the LHEP group differed with respect to the following variables: logarithm of the minimum angle of resolution best-corrected visual acuity (0.13 ± 0.13 vs. 0.25 ± 0.17; P < 0.001), central foveal thickness (218.74 ± 52.4 μm vs. 187.28 ± 50.29 μm; P = 0.008), FAF diameter (400.78 ± 189.36 μm vs. 503.37 ± 214.25 μm; P = 0.014), outer plexiform layer (OPL) diameter (382.10 ± 157.34 μm vs. 550.79 ± 228.05 μm; P = 0.0001), and disruption of external limiting membrane and ellipsoid zone, which was noted in only 1 and 3 eyes with ERM alone, respectively, and in 18 and 23 eyes with LHEP, respectively (P < 0.0001 for both observations). No difference was found for diameters measured at the level of the inner limiting membrane and schisis/cavitation. In both the ERM-alone group and the LHEP group, a strong correlation was found between the diameters measured on B-FAF and diameters measured at the OPL level on OCT images (P < 0.0001 for both groups)., Conclusions: In eyes with LMHs, a strong correlation exists between the diameters of the holes measured with B-FAF and those measured at the OPL level with OCT. This may indicate that the loss or displacement of retinal cells containing macular pigment at the OPL level, specifically photoreceptors and/or Müller cells, is involved in this vitreomaculopathy.

Purpose: We analyzed by swept-source anterior segment optical coherence tomography (SS-ASOCT) the three-dimensional iris morphology in a Caucasian population, and correlated the findings with iris color, iris sectors, subject age, and sex., Methods: One eye each from consecutive healthy emmetropic (refractive spherical equivalent ± 3 diopters) volunteers were selected for the study. The enrolled eye underwent standardized anterior segment photography to assess iris color. Iris images were assessed by SS-ASOCT for volume, thickness, width, and pupil size. Sectoral variations of morphometric data among the superior, nasal, inferior, and temporal sectors were recorded., Results: A total of 135 eyes from 57 males and 78 females, age 49 ± 17 years, fulfilled the inclusion criteria. All iris morphometric parameters varied significantly among the different sectors (all P < 0.0001). Iris total volume and thickness were significantly correlated with increasingly darker pigmentation (P < 0.0001, P = 0.0384, respectively). Neither width nor pupil diameter was influenced by iris color. Age did not affect iris volume or thickness; iris width increased and pupil diameter decreased with age (rs = 0.52, rs = -0.58, respectively). There was no effect of sex on iris volume, thickness, or pupil diameter; iris width was significantly greater in males (P = 0.007)., Conclusions: Morphology of the iris varied by iris sector, and iris color was associated with differences in iris volume and thickness. Morphological parameter variations associated with iris color, sector, age, and sex can be used to identify pathological changes in suspect eyes. To be effective in clinical settings, construction of iris morphological databases for different ethnic and racial populations is essential.

Purpose: To evaluate differences between the retinal nerve fiber layer (RNFL) thickness and RNFL + ganglion cell layer (GCL) thickness in patients affected by Alzheimer's disease (AD) and healthy patients using spectral-domain optical coherence tomography (SD-OCT)., Methods: This was a case-control prospective study. Twenty-one AD patients and 21 healthy subjects underwent neurological examination, clock-drawing test (CDT), Mini Mental State Examination (MMSE), and comprehensive ophthalmic evaluation with visual acuity. SD-OCT examination was performed using Spectralis and RTVue-100. An RNFL thickness map was obtained using the Spectralis volume protocol with 19 lines on the 30° field centered on the macula. On each B-scan, the outer RNFL limit was manually set. Statistical analysis was performed to assess interoperator RNFL evaluation thickness. An RNFL+GCL thickness map was obtained using the RTVue-100 MM6 protocol. Maps were divided into the nine ETDRS subfields and each map value in every area was evaluated. A single eye from each patient was randomly chosen to perform the analysis. Differences between AD and healthy subjects were assessed., Results: The two study groups were age and sex matched. MMSE results were 19.9 ± 3.1 and 27.9 ± 1.3, respectively (P < 0.001). There was good agreement in the manual delimitation of the RNFL layer. There was a significant difference in the thickness of both the RNFL and the RNFL+GCL in all examined fields. For example, in the inferior internal subfield, the RNFL thickness was 28.1 ± 3.1 μm for AD patients and 32.6 ± 3.8 μm for healthy subjects (P < 0.001)., Conclusions: These results indicate that RNFL and RNFL+GCL thickness measurements are reduced in AD patients compared with healthy subjects. This finding may represent a useful element for the diagnosis and follow-up of this pathology.

Purpose: To assess enhanced depth imaging optical coherence tomography (EDI-OCT) visualization of deep posterior pole structures and retrobulbar tissues in myopic eyes and evaluate ocular structural elements that influence this capability., Methods: Thirty consecutive myopic eyes (>-6 diopters) from 21 patients were enrolled. Exclusion criteria included any pathological condition affecting the posterior pole. Patients underwent biometry to assess axial length, and irises were classified as darkly or lightly pigmented. EDI-OCT scans were obtained by spectral-domain OCT to image posterior pole and retrobulbar structures. Choroidal thickness was measured manually, and for eyes in which the sclera was fully visible, scleral thickness was also measured manually. The influence of central retinal thickness, axial length, refractive error, mean choroidal thickness, mean scleral thickness, and iris pigmentation on EDI-OCT visualization of structures beyond the choroid was tested., Results: Choroidal thickness was measurable in all eyes. In 11 of 30 eyes, the sclera was not completely visible (group 1). In 19 eyes, the full scleral thickness was measurable (group 2). In seven of the group 2 eyes, the full sclera was the deepest structure detected. In the remaining 11 eyes, deeper structures were visible. Choroidal thickness was the only parameter that correlated with the ability to visualize the full sclera (P < 0.001) and deeper structures (P = 0.044)., Conclusions: The full thickness of the choroid was visualized by EDI-OCT in all eyes. Full thicknesses of the sclera and retrobulbar structures were not always visible. Choroidal thickness was the only parameter that significantly correlated with EDI-OCT visualization of deeper structures.

Purpose: We compared intrasession repeatability values produced by different spectral-domain optical coherence tomography (SD-OCT) instruments when measuring macular retinal thickness from consecutive examinations., Methods: A total of 40 eyes from 23 healthy subjects and 47 eyes from 42 patients with macular edema were enrolled in the study. Subjects underwent two consecutive SD-OCT examinations using three instruments: spectralis HRA+OCT, Cirrus, and RS 3000. For the second SD-OCT examination, the scan location was aligned to the baseline exam using different strategies: RS 3000 eye-tracking (pre-acquisition), Spectralis follow-up (during acquisition), Cirrus fovea finding (postacquisition), and Cirrus macular change analysis (postacquisition). Macular retinal thickness values from the consecutive examinations were evaluated to assess repeatability of the measurements., Results: In healthy subjects all of the strategies used for scan location alignment for the second examination provided good repeatability. For instance, intraclass correlation coefficients (ICC) from the central subfield were between 0.88 (RS 3000 eye-tracking) and 0.99 (Spectralis follow-up). In subjects affected by macular edema, the results were excellent. Cirrus macular change analysis and Spectralis follow-up produced ICC values equaled 1.00 in the central subfield. Cirrus fovea finding and RS 3000 eye-tracking produced slightly lower ICC values (0.98 and 0.99, respectively) in the central subfield., Conclusions: All of the strategies for aligning consecutive SD-OCT scan locations produced repeatable retinal thickness values. The best results were obtained using the Spectralis with follow-up and Cirrus with macular change analysis.

Purpose: Fundus autofluorescence (fundus AF) changes were monitored in a mouse model of retinal detachment (RD)., Methods: RD was induced by transscleral injection of hyaluronic acid (Healon) or sterile balanced salt solution (BSS) into the subretinal space of 4-5-day-old albino Abca4 null mutant and Abca4 wild-type mice. Images acquired by confocal scanning laser ophthalmoscopy (Spectralis HRA) were correlated with spectral domain optical coherence tomography (SD-OCT), infrared reflectance (IR), fluorescence spectroscopy, and histologic analysis. Results. In the area of detached retina, multiple hyperreflective spots in IR images corresponded to punctate areas of intense autofluorescence visible in fundus AF mode. The puncta exhibited changes in fluorescence intensity with time. SD-OCT disclosed undulations of the neural retina and hyperreflectivity of the photoreceptor layer that likely corresponded to histologically visible photoreceptor cell rosettes. Fluorescence emission spectra generated using flat-mounted retina, and 488 and 561 nm excitation, were similar to that of RPE lipofuscin. With increased excitation wavelength, the emission maximum shifted towards longer wavelengths, a characteristic typical of fundus autofluorescence., Conclusions: In detached retinas, hyper-autofluorescent spots appeared to originate from photoreceptor outer segments that were arranged within retinal folds and rosettes. Consistent with this interpretation is the finding that the autofluorescence was spectroscopically similar to the bisretinoids that constitute RPE lipofuscin. Under the conditions of a RD, abnormal autofluorescence may arise from excessive production of bisretinoid by impaired photoreceptor cells.

Purpose: To evaluate differences in fluorescein angiography (FA) and indocyanine green angiography (ICGA), findings between subjects affected by Stargardt disease (STGD) and atrophic AMD., Methods: This was a consecutive, cross-sectional case series. A total of 24 eyes of 12 patients with STGD and 23 eyes of 14 patients with atrophic AMD were enrolled in the study. Patients underwent dynamic simultaneous FA and ICGA using a dual beam confocal scanning system. Images were recorded from the initial filling of choroidal and retinal vessels throughout all the phases of the angiogram. Spectral-domain optical coherence tomography (SD-OCT) and fundus autofluorescence were also executed. FA and ICGA findings in the two groups were evaluated., Results: In 92% (22/24) of eyes affected by STGD, ICGA showed hypocyanescence from the areas of atrophy, more evident in the late phases. This finding, defined as ICGA-imaged "dark atrophy," was present in only 13% (3/23) of the eyes affected by atrophic AMD. The remaining eyes in both groups showed iso- or mild hypercyanescence from the areas of atrophy. Eyes with ICGA-imaged dark atrophy, both in STGD and in atrophic AMD groups, did not show early obscuration of the choroidal vessels by FA. SD-OCT revealed morphologically intact choroid in STGD patients with ICGA-imaged dark atrophy. In atrophic AMD eyes with ICGA-imaged dark atrophy, SD-OCT revealed a severely thinned choroid., Conclusions: Hypocyanescence by ICGA from the areas of atrophy was more frequent in STGD compared with atrophic AMD. This finding, along with SD-OCT evidence of intact choroid, suggests a possible selective damage of the choriocapillaris in STGD.

Purpose: To evaluate spectral-domain optical coherence tomography (SD-OCT) findings that predict angiographic leakage in choroidal neovascularization (CNV)., Methods: SD-OCT and fluorescein angiography (FA) images of 93 eyes of 93 patients were retrospectively analyzed. All patients were previously treated with anti-vascular endothelial growth factor agents for CNV from age-related macular degeneration. FA images were analyzed to assess the presence of leakage. SD-OCT images were analyzed to identify the overall presence of fluid, as well as specific patterns of fluid presentation, including intraretinal cystic spaces (ICS), retinal pigment epithelium detachment (PED), and neurosensory detachment (NSD). The presence of ultrastructural features such as intraretinal hyperreflective flecks and the inherent reflectivity and boundary definition of the subretinal material were evaluated. Both the association and the sensitivity, specificity, and both positive and negative predictive values of SD-OCT findings compared with FA leakage were calculated., Results: A statistically significant association between SD-OCT findings and FA leakage was found for eyes that displayed fluid, NSD, intraretinal flecks, and low reflectivity or undefined boundaries from subretinal material, and not for PED or ICS. Sensitivity and specificity for SD-OCT findings were, respectively: 94% and 27% for fluid; 68% and 88% for NSD; 81% and 83% for intraretinal flecks; 63% and 92% for undefined boundaries of subretinal material; and 94% and 87% for low reflectivity from subretinal material., Conclusions: The evidence of fluid on SD-OCT is sensitive but nonspecific in identifying FA leaky CNV. The assessment of neurosensory detachment as well as other ultrastructural elements may increase the specificity of analysis.

Purpose: To analyze progressive changes of the outer retina after vitrectomy for macular hole (MH) repair., Methods: Nineteen consecutive patients underwent vitrectomy for idiopathic MH. Spectral domain optical coherence tomography (SD-OCT) examinations were performed pre- and postoperatively during follow-up visits at 1, 3, 6, 9, and 12 months. Active eye-tracking technology ensured that the same scanning location was identified each time., Results: Ten eyes showed a normal external limiting membrane (ELM) at 1 month after surgery and 15 eyes at 3 months. The ELM was already continuous in 79% of the eyes with persistent outer foveal defects during follow-up. No eyes revealed a continuous inner segment/outer segment (IS/OS) line at 1 month, only one eye at 3 months, and 10 eyes at 12 months. No eyes had a disrupted ELM with an intact IS/OS line. Foveal cysts were visible in three eyes at 1 month and in eight eyes during follow-up. The cystic space gradually filled, resulting in a continuous IS/OS line in five of these eyes. Recovery of ELM, IS/OS, and outer nuclear layer (ONL) determined most of visual acuity improvement. The ONL appeared normal in the 10 eyes with an intact IS/OS line at last follow-up. By contrast, it was disrupted in 7 of 9 eyes with a final persistent outer foveal defect., Conclusions: The ELM is the first structure to recover after MH closure. Foveal cysts may develop during follow-up, and in the presence of an intact ONL, they may gradually fill with complete recovery of the IS/OS junction.

Purpose: A relationship has been reported between the presence of peripheral neuropathy and the density and shape of corneal nerve fibers. Peripheral neuropathy is a debilitating condition that arises from many common health problems, and its presence is often confirmed with an invasive clinical test called intramuscular electromyography (EMG). In this study, the possibility of developing an alternative or adjunct test to EMG based on the appearance of nerve fibers in corneal micrographs was explored. Since corneal imaging is virtually noninvasive compared with EMG, such a test may be administered more liberally and frequently, before neuropathy symptoms occur., Methods: A software program that automatically traces nerve fibers in corneal micrographs and generates measures based on these traces was implemented. This software was applied to a database of images collected by confocal laser scanning corneal microscopy from diabetic subjects whose levels of neuropathy were measured with EMG and from healthy subjects., Results: Trends in the nerve fiber density and various measures of shape were calculated and observed, to explore the possibility of using these measures as a clinical tool for corroborating symptoms, confirming an evaluation, or evaluating risk factors for developing neuropathy., Conclusions: Preliminary statistical trends show a potential for measuring and observing neuropathy severity or for providing an objective risk measure for a patient's ensuing condition. More work is needed in the development of the measures and in their testing to prove that the measures can be made repeatable in a clinical environment.

Purpose: To report the characteristics of confocal blue reflectance imaging in type 2 idiopathic macular telangiectasia (type 2 IMT)., Methods: In a prospective observational cross-sectional study, both eyes of 33 patients with type 2 IMT were examined by means of fundus biomicroscopy, fundus photography, fluorescein angiography, and optical coherence tomography (OCT). Confocal blue reflectance (CBR) imaging was performed using a confocal scanning laser ophthalmoscope (HRA2; Heidelberg Engineering, Heidelberg, Germany). To compare the results derived from different imaging modalities, an analysis was performed using image analysis software (Heidelberg Eye Explorer; Heidelberg Engineering)., Results: CBR imaging revealed a parafoveal area of increased reflectance that was slightly larger than the area of hyperfluorescence in late-phase fluorescein angiography. The area usually encompassed an oval parafoveal area, but sectors could be spared. A parafoveal area of increased CBR was detected in 98% of eyes that showed angiographic evidence for type 2 IMT., Conclusions: CBR imaging is a new, noninvasive, and sensitive method that may contribute to differentiate type 2 IMT from other diseases. Abnormalities of macular pigment distribution and Müller cell pathology may contribute to the phenomenon of increased CBR and thus the pathophysiology of type 2 IMT.

Purpose: To describe and classify patterns of abnormal fundus autofluorescence (FAF) in eyes with early nonexudative age-related macular disease (AMD)., Methods: FAF images were recorded in eyes with early AMD by confocal scanning laser ophthalmoscopy (cSLO) with excitation at 488 nm (argon or OPSL laser) and emission above 500 or 521 nm (barrier filter). A standardized protocol for image acquisition and generation of mean images after automated alignment was applied, and routine fundus photographs were obtained. FAF images were classified by two independent observers. The kappa statistic was applied to assess intra- and interobserver variability., Results: Alterations in FAF were classified into eight phenotypic patterns including normal, minimal change, focal increased, patchy, linear, lacelike, reticular, and speckled. Areas with abnormal increased or decreased FAF signals may or may not have corresponded to funduscopically visible alterations. For intraobserver variability, kappa of observer I was 0.80 (95% confidence interval [CI]0.71-0.89) and of observer II, 0.74. (95% CI, 0.64-0.84). For interobserver variability, kappa was 0.77 (95% CI, 0.67-0.87)., Conclusions: Various phenotypic patterns of abnormal FAF can be identified with cSLO imaging. Distinct patterns may reflect heterogeneity at a cellular and molecular level in contrast to a nonspecific aging process. The results indicate that the classification system yields a relatively high degree of intra- and interobserver agreement. It may be applicable for determination of novel prognostic determinants in longitudinal natural history studies, for identification of genetic risk factors, and for monitoring of future therapeutic interventions to slow the progression of early AMD.

Purpose: Several histopathologic studies have concluded that Stargardt's disease (Fundus flavimaculatus) is associated with abnormally high levels of lipofuscin-like material in the retinal pigment epithelium. The purpose of this study was to determine whether this material has the same fluorescence characteristics as lipofuscin in vivo and whether noninvasive measurements identify a significant elevation in this material., Methods: Five patients with autosomal recessive Stargardt's disease were included in this study, as were 45 healthy controls. All patients had the angiographic dark choroid sign. The intensity and emission spectra of lipofuscin fluorescence were measured by noninvasive fundus spectrophotometry at 7 degrees temporal to the fovea., Results: The fluorescence intensities in the five patients with Stargardt's disease were significantly higher (P < 0.0001) than those observed in normal subjects of the same age. The emission spectra in the patients are similar in shape to those measured in normals, but flecks appear to shift the spectra toward shorter wavelengths., Conclusions: The spectral characteristics of the fluorophore observed in patients with Stargardt's disease are consistent with those of retinal pigment epithelial lipofuscin. These patients have abnormally high levels of lipofuscin, confirming previous histopathologic observations. Noninvasive retinal pigment epithelial lipofuscin measurements may be a useful adjunct in the diagnosis of Stargardt's disease.-F. flavimaculatus.

Purpose: To characterize the intrinsic fluorescence (autofluorescence) of the human ocular fundus with regard to its excitation and emission spectra, age relationship, retinal location, and topography, and to identify the dominant fluorophore among the fundus layers., Methods: Using a novel fundus spectrophotometer, fluorescence measurements were made at 7 degrees temporal to the fovea and at the fovea in 30 normal subjects and in 3 selected patients. Topographic measurements were made in 3 subjects. Ex vivo measurements of fluorescence of human retinal pigment epithelium (RPE) were obtained and compared to in vivo data., Results: Fundus fluorescence reveals a broad band of emission between 500 and 750 nm, a maximum of approximately 630 nm, and optimal excitation of approximately 510 nm. Exhibiting a significant increase with age, this fluorescence is highest at 7 degrees to 15 degrees from the fovea, shows a well-defined foveal minimum, and decreases toward the periphery. In vivo fluorescence spectra are consistent with those obtained ex vivo on human RPE. Measurements with short wavelength excitation are strongly influenced by ocular media absorption and reveal an additional minor fluorophore in the fovea., Conclusions: Spectral characteristics, correlation with age, topographic distribution, and retinal location between the choriocapillaris and the photoreceptors suggest that the dominant fundus fluorophore is RPE lipofuscin. The minor fluorophore is probably in the neurosensory retina but has not been identified.