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From the Department of Ophthalmic Research, Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, Ohio.
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Abstract |
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METHODS. Cryosections were cut from the maculae of unfixed human donor eyes with AMD or from age-matched control eyes. Tissues were excited at wavelengths of 364, 488, 568, and 633 nm. Emission spectra were collected with a confocal microscope equipped with a spectrophotometric detector at 10-nm wavelength intervals between 400 and 800 nm.
RESULTS. RPE lipofuscin had strong autofluorescent emissions that were excited at all wavelengths. Bruch’s membrane exhibited strong autofluorescence with an emission peak of 485 ± 5 nm when excited with 364-nm light. At 488-, 568-, and 633-nm excitations, Bruch’s membrane and sub-RPE deposits in normal eyes exhibited minimal autofluorescence. In AMD eyes, however, both the 364- and 488-nm excitation wavelengths stimulated substantial blue-green emissions from sub-RPE deposits and Bruch’s membrane, with average pixel intensities substantially exceeding that elicited in the yellow-orange range by RPE lipofuscin.
CONCLUSIONS. These data suggest that an increase in blue-green autofluorescence of Bruch’s membrane relative to the yellow-orange autofluorescence of RPE-associated lipofuscin is associated with AMD. Knowledge of these spectra will be useful in evaluating animal models of macular degenerative disease and in diagnosis of AMD, and will provide a novel signature for further analysis of the molecular entities emitting these fluorescent signatures.
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Introduction |
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Lipofuscin is a ubiquitous material present in granules in the RPE cell with a characteristic UV-excitable fluorescence8 that is accounted for in part by A2E, the product of hydrolysis of vitamin A aldehyde and phosphatidylethanolamine.9 10 11 In Stargardt’s disease, a relationship between lipofuscin accumulation and retinal degeneration is strongly supported by studies of the abcr knockout mouse.12 The recent development of the confocal scanning laser ophthalmoscope (cLSO) has greatly facilitated the study of fundus autofluorescence in humans. Studies using the cLSO and other means of measuring fundus autofluorescence have been, for the most part, limited to inherited maculopathies such as Stargardt’s disease, in which an enhanced accumulation of lipofuscin is better established,13 14 15 16 17 18 or to patients in whom AMD has progressed to geographic atrophy.19 20 21 22 23 These studies have focused primarily on fluorescent emissions that represent RPE-associated lipofuscin.24 Although some studies suggest that fundus autofluorescence may be elevated in areas peripheral to regions of geographic atrophy21 23 or in advance of atrophy,22 fundus autofluorescence measurements have not gained wide acceptance as a diagnostic tool. Although there is a growing body of evidence linking lipofuscin accumulation with RPE atrophy,4 the connection between lipofuscin and AMD remains tenuous.
Lipofuscin granules in the RPE are not the only autofluorescent entities in the posterior pole of the eye. Autofluorescence of Bruch’s membrane and drusen have also been anecdotally reported,25 26 27 although the spectrum has never been characterized. More recently, it has been observed in a systematic study of fundus autofluorescence, that the spectrum in regions with drusen is shifted toward shorter wavelengths.20 28
To better understand the individual autofluorescence spectra of sub-RPE deposits, Bruch’s membrane, and RPE lipofuscin, we took advantage of recent developments in confocal microscopy that allow the collection of emission spectra from X–Y scans of tissue sections. Using a laser scanning confocal microscope with a spectrophotometric detector, we determined that Bruch’s membrane and sub-RPE deposits had overlapping spectra that were excited by UV and blue light with fluorescent emissions in the blue-green spectrum. Furthermore, this fluorescence was increased compared with lipofuscin fluorescence in eyes of donors with AMD relative to eyes of age-matched control donors.
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Methods |
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Confocal Microscopy
Wavelength scans were performed using the 364-, 488-, 568-, and 633-nm laser lines of a laser scanning confocal microscope (TCS-SP; Leica, Deerfield, IL) equipped with a spectrophotometric detector utilizing a Hamamatsu R6357 photodetector (Hamamatsu Corp., Hamamatsu City, Japan). This detector exhibits relatively even radiant sensitivity and quantum efficiency in the 400- to 800-nm range. Data as presented are not corrected for changes in sensitivity of the detector at different wavelengths. The spectrophotometric detector used does not rely on barrier filters, and so filter characteristics did not influence the spectrum. Scans were performed using the substrate (for UV) or SP30/70 splitters (visible light), with an open pinhole, and the detector was set to advance a 10-nm window of detection in 10-nm increments between 400 and 800 nm. Gains were set individually for each field at each excitation wavelength, by using the glow over–under function to optimize collection of data across the full 8-bit scale. This was accomplished in a 10-nm window determined by narrowing the window of detection during continuous scanning to the region of maximum fluorescence. Gains were not altered through the course of the scan. Each scan was performed using frame averaging set at 4. Data were acquired in 8-bit mode starting with the 633-nm laser and progressing to shorter wavelengths in order. Lasers were set in the park position, resulting in delivery of 200 mW for the UV Ar laser (364-nm excitation) and 300 mW for the Ar and Kr lasers (488- and 568-nm excitation) to the back of the aperture lens according to the manufacturer’s specifications. The HeNe laser (633-nm excitation) was set to deliver 1 mW to the back of the aperture lens according to the manufacturer’s specifications.
Average pixel intensities for Bruch’s membrane, sub-RPE deposits, and RPE-lipofuscin were determined on computer (Metamorph software ver. 4.5; Universal Imaging, West Chester, PA, running on a Pentium III-powered computer; Intel Corp., Mountain View, CA). Numerical data were exported to a spreadsheet (Excel 97; Microsoft, Redmond, WA). The significance of differences in spectra obtained between control and AMD-affected eyes was assessed using a two-tailed, two-sample t-test, with no variance assumptions run within the software (Excel 97; Microsoft). Bruch’s membrane and sub-RPE deposits were selected for analysis based on a differential interference contrast (DIC) image of each field analyzed. Drusen and basal laminar–linear deposit were distinguished on the basis of shape. Hard and soft drusen were defined by their round contours with clearly defined borders. We could not always distinguish between soft and hard drusen by DIC imaging. Basal laminar and basal linear deposits (BLDs) were defined as a thick continuous layer of accumulations beneath the RPE. We could not distinguish basal laminar and basal linear deposits. Drusen and BLDs were not found to have different spectra and therefore were not separated in the final analysis of the data. Therefore, for the purposes of this study we referred to drusen and BLD collectively as sub-RPE deposits. Lipofuscin granules were selected by thresholding the section in each data set with the brightest RPE-lipofuscin to reveal the specific granules. Average pixel intensities for the Bruch’s membrane, sub-RPE deposits, and RPE-lipofuscin from the same fields were normalized against the strongest measurement associated with lipofuscin in each section series. To control for photobleaching, a separate set of sections were scanned sequentially as just described, progressing from 633- through 364-nm excitation, and then rescanned. This resulted in a shift of the background relative to the signal in the second scan of approximately 10% of maximum pixel intensity across the image.
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Results |
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data sets, comprising a series of two-dimensional (XY) images stacked so that the Z-coordinate is , were accumulated for emitted light in 10-nm increments from 400 to 800 nm. Each XY- data set comprises a section series that was analyzed as a whole. The peak emission generated at ex by reflection of the excitation stimulus is omitted from all section series presented where ex is 488 nm or more. Representative DIC images are shown in Figure 1 for control (Fig. 1A) and AMD (Fig. 1B) samples. All control samples contained hard drusen similar to the one shown in Figure 1A , and fields were deliberately chosen to include drusen. More variability was present in the AMD samples (Fig. 1B) . Not all fields from eyes with AMD contained drusen, although all contained some form of deposit between the RPE and Bruch’s membrane. The most common finding was BLD. BLD was absent from all fields in control eyes.
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ex of 633 nm and moved to progressively shorter wavelengths to minimize any potential for photobleaching. The emission peak (max) for Bruch’s membrane, sub-RPE deposits, and lipofuscin at each excitation wavelength are reported in Table 1
. At ex of 633 and 568 nm in both control and AMD samples (Fig. 2)
, the dominant signal emanated from RPE-associated lipofuscin. Very small differences were noted in the max associated with each of the regions examined. These differences were confined to slight blue shifts of sub-RPE deposits and Bruch’s membrane relative to lipofuscin. Because the max for these structures was within 10 nm, in effect they all had the same spectrum (Fig. 2)
. An increase in the intensities of both Bruch’s membrane and sub-RPE deposits was noted in eyes with AMD, although this difference failed to show significance in a two-sample t-test.
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ex of 488 nm, a 10- to 15-nm difference was reproducibly obtained between the spectra elicited from Bruch’s membrane and the sub-RPE deposits in comparison with lipofuscin (Fig. 3) . The difference in max (Table 1)
was identical in both control eyes and eyes with AMD.
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max for sub-RPE deposits, Bruch’s membrane, and lipofuscin at each excitation wavelength. Values are normalized to the intensity of lipofuscin at the max obtained in Table 1 . Therefore, within a sample lipofuscin intensity can vary from 100% if there is variability in max between section series or between donor eyes. Normalization of the signals in this way permits comparison of the intensity at the max of each spectrum and indicates the strongest signal and reproducibility of that signal intensity for each region and spectrum at a given ex. Quantitative comparisons of intensity among spectra derived with different ex are not valid because of differences in detector gain settings and laser power. For this reason, all comparisons were made between the spectra elicited from sub-RPE deposits, Bruch’s membrane, and lipofuscin with the same ex.
At ex of 488 nm, lipofuscin was the dominant autofluorescent entity in control eyes (Table 2)
. A substantial increase in the intensity of the Bruch’s membrane fluorescence was detected in the AMD-affected eyes when compared with lipofuscin fluorescence. In control eyes the Bruch’s membrane normalized fluorescence at max was 53% ± 13% (mean ± SEM), whereas in eyes with AMD the normalized Bruch’s membrane’s fluorescence was 112% ± 29%. Application of the t-test to these measurements resulted in P = 0.07. Despite the differences in Bruch’s membrane’s fluorescence, no difference was detected in the normalized intensity of sub-RPE deposits in control (57 ± 17%) versus AMD-affected eyes (60% ± 5%).
At ex of 364 nm, a substantial difference was found between max (Table 1
, Fig. 4
) for Bruch’s membrane and sub-RPE deposits compared with lipofuscin. In both control and AMD eyes, Bruch’s membrane exhibited a max of 485 ± 5 nm, similar to the max obtained for sub-RPE deposits. However lipofuscin had a max of 555 ± 5 nm in control eyes and 540 ± 5 nm in eyes with AMD. Thus, we could clearly delineate different spectra for Bruch’s membrane and sub-RPE deposits with respect to lipofuscin. In AMD-affected eyes, Bruch’s membrane was clearly the brightest source of fluorescence (Table 2)
excited by 364-nm light. Furthermore, a substantial difference was found in the normalized intensity of Bruch’s membrane’s fluorescence in AMD-affected versus control eyes. This difference (82% ± 9%) in control eyes versus. 154 ± 10 in eyes with AMD was significant (P = 0.03). A small difference was noted for sub-RPE deposits, as well with sub-RPE deposits: 77% ± 4% of the intensity of lipofuscin in control eyes and 96% ± 12% in AMD-affected eyes, although this difference was not significant (P = 0.37).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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485 ± 5-nm peak of fluorescence excited from Bruch’s membrane and sub-RPE deposits with 364-nm light. The finding that Bruch’s membrane and sub-RPE deposits exhibit their own fluorescence is important, in that it must contribute to the image obtained when examining fundus autofluorescence. However, the contribution to the fundus image differs from that which we describe. Although we can make statements about the ratio of emissions generated from one region (i.e. Bruch’s membrane) in comparison to another (i.e. lipofuscin), these are separated by virtue of the magnification at which we are working (x400 in all cases), and the use of radial sections of the outer eye wall. In vivo autofluorescence measurements are made in what would be an en face orientation under the microscope and without the vertical resolution necessary to separate Bruch’s membrane, sub-RPE-deposits, and lipofuscin. Furthermore, the contribution of a druse may be substantially greater if it is large and viewed en face, as opposed to in the 8-µm-thick sections that we used. Indeed, others20 28 have shown that in eyes with drusen, there is a measurable blue shift in fundus autofluorescence. Our data confirm these earlier observations and suggest that the shift is in fact due to the novel spectrum of sub-RPE deposits compared with lipofuscin. Unfortunately, because of the limited availability of donor eyes with AMD en face sections were not available for this study.
Also of interest is the finding that the fluorescence intensity of Bruch’s membrane exceeds that of lipofuscin in AMD eyes when excited with 364-nm light. This could be due to two possibilities. The first is loss of RPE cells and thinning of the cells resulting in a decrease in lipofuscin granules. This possibility however can be ruled out in the context of this study, as the regions selected for measurement included only those with RPE cells, lipofuscin granules were specifically selected as opposed to whole cells, and the use of radial-sections eliminates thinning of the RPE from consideration. The second possibility is that the blue autofluorescence of Bruch’s membrane is in fact increased in AMD-affected eyes relative to control eyes. A qualitative examination of the sections suggests that this is in fact the case. Though it is tempting to speculate on the molecular entities responsible for this fluorescence, a large number of compounds could, alone or in combination, be responsible for this spectrum.
Can this spectrum be used diagnostically? It is tempting to suggest that quantitative measurement of the Bruch’s membrane’s autofluorescence could serve as a diagnostic tool for AMD, and in fact we describe herein a novel spectrum that differs in intensity in control eyes and eyes with AMD. Unfortunately, this spectrum is excited by UV light that is blocked from entering the eye by the cornea and lens. Furthermore, in our studies the pigment granules of the RPE did not influence the delivery of light to the sample. Changing to an en face orientation would place the RPE pigment granules between Bruch’s membrane and the light source, as well as between Bruch’s membrane and a detector, potentially quenching the effect that we describe in radial section. However, the increase in fluorescence observed using 488-nm light could be put to use. Barring a damping effect of the pigment granules, the total fluorescence detected when excited by 488-nm light should be increased relative to control eyes. Earlier fundus autofluorescence studies used 510- or 470-nm excitation wavelengths.23
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In those studies, lipofuscin fluorescence was found to be more efficiently excited at 510 nm with a greater contribution derived from sub-RPE deposits when ex was 470 nm. One difference from our findings is that others have found that peak lipofuscin fluorescence in vivo exhibits a max of 620 nm as opposed to the 555 nm we observed. This could be in part due to the difference in excitation wavelength—510 nm in the former studies as opposed to 488 nm in the present study—and to macular pigment or age-associated yellowing of the lens.
In healthy eyes, we found that the max of lipofuscin in RPE cells was 555 nm when excited with light at 364 nm, similar to the 565 max reported by Sparrow et al.30
for A2E in unfixed RPE cells excited with 380-nm light. We also found that the emission spectrum of lipofuscin shifted gradually toward the red as the excitation wavelength was increased, similar to the data reported by Delori.24
The shift of max to 540 nm that we observed for lipofuscin in AMD-affected eyes may be due to overlap of RPE and sub-RPE deposits in some sections from those eyes, or could represent a real change in the spectral characteristics and thus the composition of lipofuscin in eyes with AMD.
In summary, we examined the autofluorescent spectra of Bruch’s membrane, sub-RPE deposits, and lipofuscin in AMD-affected and age-matched control eyes. We found a novel UV-excitable spectrum associated with Bruch’s membrane and sub-RPE deposits that appears to be present at higher intensities in AMD-affected eyes than in age-matched control eyes.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health Grant R01-EY13160 (ADM), a Kirchgessner Foundation research grant (ADM), the Foundation Fighting Blindness (JGH), and the Cleveland Clinic Foundation.
Submitted for publication June 19, 2001; revised March 1, 2002; accepted March 13, 2002.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Alan D. Marmorstein, Cole Eye Institute, i-31, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195; marmora{at}ccf.org.
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References |
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