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Near-Infrared Autofluorescence Imaging of the Fundus

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Near-Infrared Autofluorescence Imaging of the Fundus Near-Infrared Autofluorescence Imaging of the Fundus: Visualization of Ocular Melanin Claudia N. Keilhauer1 and Franc¸ois C. Delori2 PURPOSE. To evaluate the origin of the near-infrared autofluo- ICG angiography. They demonstrated that pseudofluorescence rescence (AF) of the fundus detected by scanning laser oph- did not substantially affect their images, but that a faint AF thalmoscopy and compare the distribution of this AF with that emanated from several pathologic structures (the normal eye of lipofuscin. was not investigated). They suggested that degradation prod- METHODS. AF [787] fundus images (excitation [Exc.] 787 nm; ucts of blood, lipofuscin deposits, and/or melanin contributed emission [Emi.] Ͼ800 nm) were recorded with a confocal to this AF. We have further investigated near-infrared AF imag- scanning laser ophthalmoscope, in 85 normal subjects (ages: ing using the superior imaging modality provided by the con- 11–77 years) and in 25 patients with AMD and other retinal focal SLO, in conjunction with the same excitation wavelength diseases. Standard AF [488] images (Exc. 488 nm; Emi. Ͼ500 (787 nm) and detection system that is normally used for ICG nm) were recorded in a subset of the population. angiography (Keilhauer CN, et al. IOVS 2005;46:ARVO E-Ab- stract 1394; Weinberger AWA, et al. IOVS 2005;46:ARVO E-Ab- RESULTS. The fovea exhibits higher AF[787] than the perifovea ϳ stract 2585). In this study, we demonstrate that normal eyes in an area 8° in diameter, roughly equivalent to the area of exhibit a characteristic near-infrared AF distribution and have higher RPE melanin seen in AF[488] and color images. The Ͻ investigated the origin of this AF, in a population of subjects ratio of foveal to perifoveal AF[787] decreases with age (P with no retinal disease and in selected clinical cases. 0.0001) and is higher in subjects with light irides (P ϭ 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 METHODS geographic atrophy particularly in subjects with light irides. Population CONCLUSIONS. AF[787] originates from the RPE and to a varying degree from the choroid. Oxidized melanin, or compounds NIR AF images were obtained in 85 subjects with normal retinal status closely associated with melanin, contributes substantially to (49 women and 36 men; mean age: 47 Ϯ 18 years; range, 11–77 years). this AF, but other fluorophores cannot be excluded at this Iris colors were light (blue, gray) in 34 subjects and dark (green, hazel, stage. Confocal AF[787] imaging may provide a new modality brown) in 51 subjects. Two subjects were black Africans. Images were to visualize pathologic features of the RPE and the choroid, acquired from one eye in 49 subjects and from both eyes in 36 and, together with AF[488] imaging, offers a new tool to study subjects; interocular correspondence was assessed, and one eye was biological changes associated with aging of the RPE and selected randomly for other analyses. Images of fundus diseases have pathology. (Invest Ophthalmol Vis Sci. 2006;47:3556–3564) also been analyzed to differentiate AF contributions from fundus layers DOI:10.1167/iovs.06-0122 or to illustrate the AF of various pigments. These diseases were peri- papillary atrophy (n ϭ 3), hyperpigmentation (n ϭ 8), geographic utofluorescence (AF) imaging is playing an increasingly atrophy (n ϭ 11, ages: 69–81 years) in AMD, and macular hole (n ϭ 3). Aimportant role in the diagnosis of age-related macular de- The tenets of the Declaration of Helsinki were observed. The generation (AMD) and retinal dystrophies. Fundus AF gener- Institutional Review Board of the Eye Clinic (University of Wu¨rzburg) ated with short-wavelength excitation is dominated by RPE granted approval for this project. Informed consent was obtained from lipofuscin,1 a complex mixture of fluorophores that are by- all subjects. products of the visual cycle,2,3 and accumulate in the RPE after 4 phagocytosis. Confocal scanning laser ophthalmoscopy (SLO) Retinal Imaging has made this imaging modality accessible as a clinical tool.5–7 An important advantage of AF imaging has been that the signal NIR AF-images—AF[787]—were recorded with a confocal scanning originates principally from the RPE, resulting in a relatively laser ophthalmoscope (HRA, Retinal Angiograph; Heidelberg Engineer- simple interpretation of the images. Fundus autofluorescence ing, Heidelberg, Germany). Laser diode excitation was at 787 Ϯ 2nm excited in the near-infrared (NIR) at 805 nm was first reported (power at the pupil: 1.9 mW) and the detection filter transmitted light by Piccolino et al.8 using a nonconfocal video-imaging system above 800 nm (filter rejection: described later). The field was 30° ϫ as part of an investigation of possible pseudofluorescence in 30° (512 ϫ 512 pixels), and always included the optic disc and the macula (the foveola was at least 100 pixels from the image edge). The confocal depth of the camera was ϳ1100 ␮m (measured by moving an From the 1Department of Ophthalmology, University Hospital, NIR-fluorescent retina in an artificial eye through the focal plane and Wu¨rzburg, Germany; the 2Schepens Eye Research Institute, Harvard measuring the locations were the returned signal is at half maximum). Medical School, Boston, Massachusetts. This field size is large enough to collect light simultaneously from the Supported in part by National Eye Institute Grant EY08511 (FCD). retina and choroid, while rejecting light originating from the lens and Submitted for publication February 3, 2006; revised April 12, from a large part of the vitreous. 2006; accepted June 12, 2006. All images were acquired by the same operator (CNK) for eyes with Disclosure: C.N. Keilhauer, None; F.C. Delori, None dilated pupils. Focusing was achieved at 815 nm, and reflectance The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- images were acquired. After a switch to the 787-nm excitation (ICG ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. mode), the sensitivity was increased until the vessels and the disc Corresponding author: Claudia N. Keilhauer, Department of Oph- appeared as faint features and ϳ40 images were acquired. In 38 study thalmology, University Eye Hospital Wu¨rzburg, Josef-Schneider Strasse subjects (mean age: 45 Ϯ 21 years), we also acquired AF[488] images 11, Wu¨rzburg, Germany; [email protected]. (excitation: 488 nm; power: 270 ␮W). Investigative Ophthalmology & Visual Science, August 2006, Vol. 47, No. 8 3556 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 10/01/2021 IOVS, August 2006, Vol. 47, No. 8 Autofluorescence Imaging of Ocular Melanin 3557 FIGURE 1. AF[787] images in five subjects with normal retinal status. Ages and iris color (L: light; D: dark) as indicated. AF[488] images are shown for comparison in (A) and (E). Bars and plus signs indicate the po- sition of the fovea as defined by the darkest point of MP distribution in the AF[488] image. The square (1.25° ϫ 1.25°) and rectangles (2.50° ϫ 1.25°) in (A) show the areas in which mean GLs were measured on both AF[488] and AF[787] images; the three perifoveal sites are at the same distance from the fovea, and the nasal site is midway between the fovea and the disc center. A temporal site was not used because it often was partially outside the image. All AF[787] images showed an area of high IR fluorescence which corre- sponded to the area of higher mela- nin pigmentation in the AF[488] im- ages, extending outside the densest MP distribution (A, E). Images (B) and (C) are from subjects with the largest and smallest contrast of the bright area in our population. The fovea location in (D) is marked by a local reduction in AF. Choroidal ves- sels in (C) and (D) are delineated against the brighter AF originating from the outer choroid. A nevus (E, arrows) exhibits bright AF[787] but without marked increase of lipofus- cin in the RPE, as demonstrated by the AF[488]. The GL on the nevus was 1.9 times higher than the neigh- boring area. Image Analysis at a black screen in the dark (zero GL varied from 9 to 23, because of electronic variations). Only images with mean exposures at the three Eighteen AF[787] images were selected for highest exposure and peripheral sites Ͼ2 GLs above zero were included for analysis. absence of severe eye movements, aligned, and averaged (nine images were used for AF[488] and one for NIR reflectance). All images shown Filter Rejection herein have been histogram stretched. For quantitative analysis of the AF distribution, we used nonstretched images in conjunction with Because AF[787] signals were very low, it was important to assess IGOR image analysis software (WaveMetrics, Lake Oswego, OR). Mean whether filter rejection was adequate. We first determined the filters’ gray levels (GL) were measured at the center of the fovea and at three rejection by acquiring images of the reflection of a coverslip (placed at perifoveal sites (Fig. 1A). GLs from different images cannot be com- ϳ20 cm from the camera) obtained (1) in the AF[787]-mode (power: pared with each other (different sensitivities, laser powers, and pupil 1.9 mW; sensitivity adjusted to show the leak), (2) in the 815 nm- diameters), but ratios of GL from the same image can be used to assess reflectance-mode (power: 63 ␮W; same electronic sensitivity) with AF distribution. We defined the zero GL as the mean GL of the 2500 insertion of a glass neutral-density filter (NDF) placed at 45° to the least-exposed pixels of each image or ϳ1% of the image area (histo- camera axis (to avoid reflections from the NDF) between the coverslip gram analysis).
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