Quantifying Fundus Autofluorescence in Patients With Retinitis Pigmentosa.

Purpose: Using quantitative fundus autofluorescence (qAF), we analyzed short-wavelength autofluorescent (SW-AF) rings in RP. Methods: Short-wavelength autofluorescent images (486 nm excitation) of 40 patients with RP (69 eyes) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference. Mean qAF was measured in eight preset segments (qAF8) and in region of interest (ROI)-qAF (200-700 µm) within and external to the borders of the rings at superior, temporal, and inferior sites relative to the ring. For both groups, qAF in patients with RP was compared to age-similar and race/ethnicity-matched healthy eyes at equivalent retinal locations. Results: In 71% of eyes of RP patients, qAF8 acquired internal to the inner border of the ring, was within the 95% confidence interval (CI) for healthy eyes, while in the remaining RP eyes qAF8 was either higher or lower than the CI. Measured external to the ring, qAF8 values were within the CI in 47% of RP eyes with the other eyes being higher or lower. In 28% of sites measured by ROI-qAF within the SW-AF ring, values were above the 95% CI of healthy controls. Region of interest-qAF measured just external to the ring was within the CI of healthy eyes in 74% of locations. The average local elevation in qAF within the ring was approximately 15%. In SD-OCT scans, photoreceptor-attributable reflectivity bands were thinned within and external to the ring. Conclusions: Increased fluorophore production may be a factor in the formation of the SW-AF rings in RP.

Quantitative Fundus Autofluorescence in Best Vitelliform Macular Dystrophy: RPE Lipofuscin is not Increased in Non-Lesion Areas of Retina.

Since the lipofuscin of retinal pigment epithelial (RPE) cells has been implicated in the pathogenesis of Best vitelliform macular dystrophy, we quantified fundus autofluorescence (quantitative fundus autofluorescence, qAF) as an indirect measure of RPE lipofuscin levels. Mean non-lesion qAF was found to be within normal limits for age. By spectral domain optical coherence tomography (SD-OCT) vitelliform lesions presented as fluid-filled subretinal detachments containing reflective material. We discuss photoreceptor outer segment debris as the source of the intense fluorescence of these lesions and loss of anion channel functioning as an explanation for the bullous photoreceptor-RPE detachment. Unexplained is the propensity of the disease for central retina.

Quantitative Fundus Autofluorescence and Optical Coherence Tomography in ABCA4 Carriers.

PURPOSE: To assess whether carriers of ABCA4 mutations have increased RPE lipofuscin levels based on quantitative fundus autofluorescence (qAF) and whether spectral-domain optical coherence tomography (SD-OCT) reveals structural abnormalities in this cohort. METHODS: Seventy-five individuals who are heterozygous for ABCA4 mutations (mean age, 47.3 years; range, 9-82 years) were recruited as family members of affected patients from 46 unrelated families. For comparison, 57 affected family members with biallelic ABCA4 mutations (mean age, 23.4 years; range, 6-67 years) and two noncarrier siblings were also enrolled. Autofluorescence images (30°, 488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference. The gray levels (GLs) of each image were calibrated to the reference, zero GL, magnification, and normative optical media density to yield qAF. Horizontal SD-OCT scans through the fovea were obtained and the thicknesses of the outer retinal layers were measured. RESULTS: In 60 of 65 carriers of ABCA4 mutations (age range, 9-60), qAF levels were within normal limits (95% confidence level) observed for healthy noncarrier subjects, while qAF levels of affected family members were significantly increased. Perifoveal fleck-like abnormalities were observed in fundus AF images in four carriers, and corresponding changes were detected in the outer retinal layers in SD-OCT scans. Thicknesses of the outer retinal layers were within the normal range. CONCLUSIONS: With few exceptions, individuals heterozygous for ABCA4 mutations and between the ages of 9 and 60 years do not present with elevated qAF. In a small number of carriers, perifoveal fleck-like changes were visible.

Quantitative Fundus Autofluorescence and Optical Coherence Tomography in PRPH2/RDS- and ABCA4-Associated Disease Exhibiting Phenotypic Overlap.

PURPOSE: To assess whether quantitative fundus autofluorescence (qAF), a measure of RPE lipofuscin, and spectral-domain optical coherence tomography (SD-OCT) can aid in the differentiation of patients with fundus features that could either be related to ABCA4 mutations or be part of the phenotypic spectrum of pattern dystrophies. METHODS: Autofluorescence images (30°, 488-nm excitation) from 39 patients (67 eyes) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference and were quantified as previously described. In addition, horizontal SD-OCT images through the fovea were obtained. Patients were screened for ABCA4 and PRPH2/RDS mutations. RESULTS: ABCA4 mutations were identified in 19 patients (mean age, 37 ± 12 years) and PRPH2/RDS mutations in 8 patients (mean age, 48 ± 13 years); no known ABCA4 or PRPH2/RDS mutations were found in 12 patients (mean age, 48 ± 9 years). Differentiation of the groups using phenotypic SD-OCT and AF features (e.g., peripapillary sparing, foveal sparing) was not reliable. However, patients with ABCA4 mutations could be discriminated reasonably well from other patients when qAF values were corrected for age and race. In general, ABCA4 patients had higher qAF values than PRPH2/RDS patients, while most patients without mutations in PRPH2/RDS or ABCA4 had qAF levels within the normal range. CONCLUSIONS: The high qAF levels of ABCA4-positive patients are a hallmark of ABCA4-related disease. The reason for high qAF among many PRPH2/RDS-positive patients is not known; higher RPE lipofuscin accumulation may be a primary or secondary effect of the PRPH2/RDS mutation.

Quantitative fundus autofluorescence distinguishes ABCA4-associated and non-ABCA4-associated bull's-eye maculopathy.

PURPOSE: Quantitative fundus autofluorescence (qAF) and spectral-domain optical coherence tomography (SD OCT) were performed in patients with bull's-eye maculopathy (BEM) to identify phenotypic markers that can aid in the differentiation of ABCA4-associated and non-ABCA4-associated disease. DESIGN: Prospective cross-sectional study at an academic referral center. SUBJECTS: Thirty-seven BEM patients (age range, 8-60 years) were studied. All patients exhibited a localized macular lesion exhibiting a smooth contour and qualitatively normal-appearing surrounding retina without flecks. Control values consisted of previously published data from 277 healthy subjects (374 eyes; age range, 5-60 years) without a family history of retinal dystrophy. METHODS: Autofluorescence (AF) images (30°, 488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The grey levels (GLs) from 8 circularly arranged segments positioned at an eccentricity of approximately 7° to 9° in each image were calibrated to the reference (0 GL), magnification, and normative optical media density to yield qAF. In addition, horizontal SD OCT images through the fovea were obtained. All patients were screened for ABCA4 mutations using the ABCR600 microarray, next-generation sequencing, or both. MAIN OUTCOME MEASURES: Quantitative AF, correlations between AF and SD OCT, and genotyping for ABCA4 variants. RESULTS: ABCA4 mutations were identified in 22 patients, who tended to be younger (mean age, 21.9±8.3 years) than patients without ABCA4 mutations (mean age, 42.1±14.9 years). Whereas phenotypic differences were not obvious on the basis of qualitative fundus AF and SD OCT imaging, with qAF, the 2 groups of patients were clearly distinguishable. In the ABCA4-positive group, 37 of 41 eyes (19 of 22 patients) had qAF8 of more than the 95% confidence interval for age. Conversely, in the ABCA4-negative group, 22 of 26 eyes (13 of 15 patients) had qAF8 within the normal range. CONCLUSIONS: The qAF method can differentiate between ABCA4-associated and non-ABCA4-associated BEM and may guide clinical diagnosis and genetic testing.

Quantitative fundus autofluorescence in recessive Stargardt disease.

PURPOSE: To quantify fundus autofluorescence (qAF) in patients with recessive Stargardt disease (STGD1). METHODS: A total of 42 STGD1 patients (ages: 7-52 years) with at least one confirmed disease-associated ABCA4 mutation were studied. Fundus AF images (488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope 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, zero GL, magnification, and normative optical media density to yield qAF. Texture factor (TF) was calculated to characterize inhomogeneities in the AF image and patients were assigned to the phenotypes of Fishman I through III. RESULTS: Quantified fundus autofluorescence in 36 of 42 patients and TF in 27 of 42 patients were above normal limits for age. Young patients exhibited the relatively highest qAF, with levels up to 8-fold higher than healthy eyes. Quantified fundus autofluorescence and TF were higher in Fishman II and III than Fishman I, who had higher qAF and TF than healthy eyes. Patients carrying the G1916E mutation had lower qAF and TF than most other patients, even in the presence of a second allele associated with severe disease. CONCLUSIONS: Quantified fundus autofluorescence is an indirect approach to measuring RPE lipofuscin in vivo. We report that ABCA4 mutations cause significantly elevated qAF, consistent with previous reports indicating that increased RPE lipofuscin is a hallmark of STGD1. Even when qualitative differences in fundus AF images are not evident, qAF can elucidate phenotypic variation. Quantified fundus autofluorescence will serve to establish genotype-phenotype correlations and as an outcome measure in clinical trials.

Quantitative fundus autofluorescence and optical coherence tomography in best vitelliform macular dystrophy.

PURPOSE: Quantitative fundus autofluorescence (qAF), spectral domain optical coherence tomography (SD-OCT) segmentation, and multimodal imaging were performed to elucidate the pathogenesis of Best vitelliform macular dystrophy (BVMD) and to identify abnormalities in lesion versus nonlesion fundus areas. METHODS: Sixteen patients with a clinical diagnosis of BVMD were studied. Autofluorescence images (30°, 488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The grey levels (GLs) of each image were calibrated to the reference, zero GL, magnification, and normative optical media density, to yield qAF. Horizontal SD-OCT scans were obtained and retinal layers manually segmented. Additionally, color and near-infrared reflectance (NIR-R) images were registered to AF images. All patients were screened for mutations in BEST1. In three additional BVMD patients, in vivo spectrofluorometric measurements were obtained within the vitelliform lesion. RESULTS: Mean nonlesion qAF was within normal limits for age. Maximum qAF within the lesion was markedly increased compared with controls. By SD-OCT segmentation, outer segment equivalent thickness was increased and outer nuclear layer thickness decreased in the lesion. Changes were also present in a transition zone beyond the lesion border. In subclinical patients, no abnormalities in retinal layer thickness were identified. Fluorescence spectra recorded from the vitelliform lesion were consistent with those of retinal pigment epithelial cell lipofuscin. CONCLUSIONS: Based on qAF, mutations in BEST1 do not cause increased lipofuscin levels in nonlesion fundus areas.

Quantitative fundus autofluorescence in healthy eyes.

PURPOSE: Fundus autofluorescence was quantified (qAF) in subjects with healthy retinae using a standardized approach. The objective was to establish normative data and identify factors that influence the accumulation of RPE lipofuscin and/or modulate the observed AF signal in fundus images. METHODS: AF images were acquired from 277 healthy subjects (age range: 5-60 years) by employing a Spectralis confocal scanning laser ophthalmoscope (cSLO; 488-nm excitation; 30°) equipped with an internal fluorescent reference. For each image, mean gray level was calculated as the average of eight preset regions, and was calibrated to the reference, zero-laser light, magnification, and optical media density from normative data on lens transmission spectra. Relationships between qAF and age, sex, race/ethnicity, eye color, refraction/axial length, and smoking status were evaluated as was measurement repeatability and the qAF spatial distribution. RESULTS: qAF levels exhibited a significant increase with age. qAF increased with increasing eccentricity up to 10° to 15° from the fovea and was highest superotemporally. qAF values were significantly greater in females, and, compared with Hispanics, qAF was significantly higher in whites and lower in blacks and Asians. No associations with axial length and smoking were observed. For two operators, between-session repeatability was ± 9% and ± 12%. Agreement between the operators was ± 13%. CONCLUSIONS: Normative qAF data are a reference tool essential to the interpretation of qAF measurements in ocular disease.

Quantitative fundus autofluorescence in mice: correlation with HPLC quantitation of RPE lipofuscin and measurement of retina outer nuclear layer thickness.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Visualization of the optic fissure in short-wavelength autofluorescence images of the fundus.

PURPOSE: To document and explain the presence, inferior to the optic disc, of a distinct vertical boundary between two retinal areas of different short-wavelength autofluorescence (SW-AF) intensities. METHODS: SW-AF images of the inferonasal region were acquired from 32 healthy subjects. Additionally, color, 488-nm reflectance (488-R), near-infrared reflectance (NIR-R), NIR autofluorescence (NIR-AF) images, and a spectral domain optical coherence tomography (SD-OCT) image were obtained in selected subjects. Gray levels (GL) on both sides of the demarcation line were measured in SW-AF and 488-R at fixed distances from the disc center. RESULTS: A curved demarcation line inferior to the optic disc was observed on SW-AF images in 31/32 subjects. AF levels on the nasal side were 13% (±6%) lower than on the temporal side at 20° inferior to the disc center. The contrast between the nasal and the temporal areas was not significantly affected by age, sex, refractive error, race, or iris color. The demarcation line visible in SW-AF was also seen, though with reduced contrast, in approximately 80% of the 488-R images (lower reflectance on the nasal side) and 50% of color images. The boundary was not detected by NIR-R, NIR-AF, or by SD-OCT imaging. CONCLUSIONS: The location and the distinctness of the demarcation line may indicate a relationship to the closed embryonic optic fissure. The reduced SW-AF intensity and 488-R reflectance observed on the nasal side of this line may be attributable to lower lipofuscin and melanin content per unit area, possibly resulting from a difference in RPE cell shape.

Optimization of in vivo confocal autofluorescence imaging of the ocular fundus in mice and its application to models of human retinal degeneration.

PURPOSE: To investigate the feasibility and to identify sources of experimental variability of quantitative and qualitative fundus autofluorescence (AF) assessment in mice. METHODS: Blue (488 nm) and near-infrared (790 nm) fundus AF imaging was performed in various mouse strains and disease models (129S2, C57Bl/6, Abca4(-/-), C3H-Pde6b(rd1/rd1), Rho(-/-), and BALB/c mice) using a commercially available scanning laser ophthalmoscope. Gray-level analysis was used to explore factors influencing fundus AF measurements. RESULTS: A contact lens avoided cataract development and resulted in consistent fundus AF recordings. Fundus illumination and magnification were sensitive to changes of the camera position. Standardized adjustment of the recorded confocal plane and consideration of the pupil area allowed reproducible recording of fundus AF from the retinal pigment epithelium with an intersession coefficient of repeatability of ±22%. Photopigment bleaching occurred during the first 1.5 seconds of exposure to 488 nm blue light (∼10 mW/cm(2)), resulting in an increase of fundus AF. In addition, there was a slight decrease in fundus AF during prolonged blue light exposure. Fundus AF at 488 nm was low in animals with an absence of a normal visual cycle, and high in BALB/c and Abca4(-/-) mice. Degenerative alterations in Pde6b(rd1/rd1) and Rho(-/-) were reminiscent of findings in human retinal disease. CONCLUSIONS: Investigation of retinal phenotypes in mice is possible in vivo using standardized fundus AF imaging. Correlation with postmortem analysis is likely to lead to further understanding of human disease phenotypes and of retinal degenerations in general. Fundus AF imaging may be useful as an outcome measure in preclinical trials, such as for monitoring effects aimed at lowering lipofuscin accumulation in the retinal pigment epithelium.

Quantification of fluorescein-stained drusen associated with age-related macular degeneration.

BACKGROUND: Previous studies of age-related macular degeneration have not quantified the number of drusen that accumulate fluorescein. Histopathologic studies have demonstrated druse subregions with different degrees of hydrophobicity, and these subregions might potentially exhibit different degrees of fluorescein uptake. METHODS: We evaluated macular drusen from 35 age-related macular degeneration patients by measuring druse area in color digital images and fluorescein angiograms, using 2 morphometric methods. RESULTS: Of 828 drusen evaluated, 405 had a corresponding fluorescein angiogram signal. About half of all drusen per eye (49.57%) stained in each participant. Among fluorescein-stained drusen, druse size measured in color images did not differ significantly from the sizes measured in corresponding fluorescein images (P = 0.8105), across the range of druse sizes. CONCLUSION: These findings indicate that our understanding of drusen subregion staining may not directly correlate to in vivo observations of macular drusen in age-related macular degeneration.

Vitamin A activates rhodopsin and sensitizes it to ultraviolet light.

The visual pigment, rhodopsin, consists of opsin protein with 11-cis retinal chromophore, covalently bound. Light activates rhodopsin by isomerizing the chromophore to the all-trans conformation. The activated rhodopsin sets in motion a biochemical cascade that evokes an electrical response by the photoreceptor. All-trans retinal is eventually released from the opsin and reduced to vitamin A. Rod and cone photoreceptors contain vast amounts of rhodopsin, so after exposure to bright light, the concentration of vitamin A can reach relatively high levels within their outer segments. Since a retinal analog, ß-ionone, is capable of activating some types of visual pigments, we tested whether vitamin A might produce a similar effect. In single-cell recordings from isolated dark-adapted salamander green-sensitive rods, exogenously applied vitamin A decreased circulating current and flash sensitivity and accelerated flash response kinetics. These changes resembled those produced by exposure of rods to steady light. Microspectrophotometric measurements showed that vitamin A accumulated in the outer segments and binding of vitamin A to rhodopsin was confirmed in in vitro assays. In addition, vitamin A improved the sensitivity of photoreceptors to ultraviolet (UV) light. Apparently, the energy of a UV photon absorbed by vitamin A transferred by a radiationless process to the 11-cis retinal chromophore of rhodopsin, which subsequently isomerized. Therefore, our results suggest that vitamin A binds to rhodopsin at an allosteric binding site distinct from the chromophore binding pocket for 11-cis retinal to activate the rhodopsin, and that it serves as a sensitizing chromophore for UV light.

The value of measurement of macular carotenoid pigment optical densities and distributions in age-related macular degeneration and other retinal disorders.

There is increasing recognition that the optical and antioxidant properties of the xanthophyll carotenoids lutein and zeaxanthin play an important role in maintaining the health and function of the human macula. In this review article, we assess the value of non-invasive quantification of macular pigment levels and distributions to identify individuals potentially at risk for visual disability or catastrophic vision loss from age-related macular degeneration, and we consider the strengths and weaknesses of the diverse measurement methods currently available.

Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium.

PURPOSE: Autofluorescence fundus imaging using an adaptive optics scanning laser ophthalmoscope (AOSLO) allows for imaging of individual retinal pigment epithelial (RPE) cells in vivo. In this study, the potential of retinal damage was investigated by using radiant exposure levels that are 2 to 150 times those used for routine imaging. METHODS: Macaque retinas were imaged in vivo with a fluorescence AOSLO. The retina was exposed to 568- or 830-nm light for 15 minutes at various intensities over a square (1/2) degrees per side. Pre- and immediate postexposure images of the photoreceptors and RPE cells were taken over a 2 degrees field. Long-term AOSLO imaging was performed intermittently from 5 to 165 days after exposure. Exposures delivered over a uniform field were also investigated. RESULTS: Exposures to 568-nm light caused an immediate decrease in autofluorescence of RPE cells. Follow-up imaging revealed either full recovery of autofluorescence or long-term damage in the RPE cells at the exposure. The outcomes of AOSLO exposures and uniform field exposures of equal average power were not significantly different. No effects from 830-nm exposures were observed. CONCLUSIONS: The study revealed a novel change in RPE autofluorescence induced by 568-nm light exposure. Retinal damage occurred as a direct result of total average power, independent of the light-delivery METHOD: Because the exposures were near or below permissible levels in laser safety standards, these results suggest that caution should be used with exposure of the retina to visible light and that the safety standards should be re-evaluated for these exposure conditions.

Maximum permissible exposures for ocular safety (ANSI 2000), with emphasis on ophthalmic devices.

After discussing the rationale and assumptions of the ANSI Z136.1-2000 Standard for protection of the human eye from laser exposure, we present the concise formulation of the exposure limits expressed as maximum permissible radiant exposure (in J/cm(2)) for light overfilling the pupil. We then translate the Standard to a form that is more practical for typical ophthalmic devices or in vision research situations, implementing the special qualifications of the Standard. The safety limits are then expressed as radiant power (watts) entering the pupil of the eye. Exposure by repetitive pulses is also addressed, as this is frequently employed in ophthalmic applications. Examples are given that will familiarize potential users with this format.

Near-infrared autofluorescence imaging of the fundus: visualization of ocular melanin.

PURPOSE: To evaluate the origin of the near-infrared autofluorescence (AF) of the fundus detected by scanning laser ophthalmoscopy and compare the distribution of this AF with that of lipofuscin. METHODS: AF [787] fundus images (excitation [Exc.] 787 nm; emission [Emi.] >800 nm) were recorded with a confocal scanning laser ophthalmoscope, in 85 normal subjects (ages: 11-77 years) and in 25 patients with AMD and other retinal diseases. Standard AF [488] images (Exc. 488 nm; Emi. >500 nm) were recorded in a subset of the population. RESULTS: The fovea exhibits higher AF[787] than the perifovea in an area approximately 8 degrees in diameter, roughly equivalent to the area of higher RPE melanin seen in AF[488] and color images. The ratio of foveal to perifoveal AF[787] decreases with age (P < 0.0001) and is higher in subjects with light irides (P = 0.04). Higher AF[787] emanates from hyperpigmentation, from the choroidal pigment (nevi, outer layers) and from the pigment epithelium and stroma of the iris. Low AF[787] is observed in geographic atrophy particularly in subjects with light irides. CONCLUSIONS: AF[787] originates from the RPE and to a varying degree from the choroid. Oxidized melanin, or compounds closely associated with melanin, contributes substantially to this AF, but other fluorophores cannot be excluded at this stage. Confocal AF[787] imaging may provide a new modality to visualize pathologic features of the RPE and the choroid, and, together with AF[488] imaging, offers a new tool to study biological changes associated with aging of the RPE and pathology.

Bimodal spatial distribution of macular pigment: evidence of a gender relationship.

The spatial distribution of the optical density of the human macular pigment measured by two-wavelength autofluorescence imaging exhibits in over half of the subjects an annulus of higher density superimposed on a central exponential-like distribution. This annulus is located at about 0.7 degrees from the fovea. Women have broader distributions than men, and they are more likely to exhibit this bimodal distribution. Maxwell's spot reported by subjects matches the measured distribution of their pigment. Evidence that the shape of the foveal depression may be gender related leads us to hypothesize that differences in macular pigment distribution are related to anatomical differences in the shape of the foveal depression.

Autofluorescence method to measure macular pigment optical densities fluorometry and autofluorescence imaging.

Non-invasive measurement of the optical density of the human macular pigment by the autofluorescence method takes advantage of the fluorescence of lipofuscin in the human retinal pigment epithelium. Measuring the intensity of fluorescence above 550 nm, where macular pigment has essentially zero absorption, and stimulating the fluorescence with two wavelengths, one well absorbed by macular pigment and the other minimally absorbed by macular pigment, provides a single-pass measurement of the macular pigment optical density. The method is implemented either by fluorometry of lipofuscin to yield the optical density of the macular pigment in a 2 degrees diameter central area, or by autofluorescence imaging to yield a high-resolution map of the macular pigment distribution.