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F.C. Delori, D.G. Goger, J.R. Sparrow; Spectral Characteristics of Lipofuscin Autofluorescence in RPE Cells of Donor Eyes . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1715.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Fundus autofluorescence (AF) has been shown to originate principally from RPE lipofuscin (LF), a collection of fluorophores of which A2E is a dominant and deleterious component. We compared the spectral properties of lipofuscin-containing RPE isolated from human donor eyes (LF-RPE) with the previously measured spectra from ARPE-19 cells (devoid of native LF) which accumulated A2E in culture (A2E-RPE). Methods: RPE harvested from human donor eyes (6 eyes aged 67-80 years) was centrifuged onto polylysine-coated slides; the monolayers were fixed immediately. Sheets of RPE were also isolated from 2 fetal eyes, one of which was measured in its native state, the other after A2E accumulation. AF spectra were measured for 8 excitation wavelengths between 430 and 570 nm (HW: 20 nm). Results: Emission spectra from LF-RPE are slightly broader (HW=180 nm) than those of A2E-RPE (HW=160 nm). For excitation at 470 nm, peak emission occurs at 605±9 and 600±4 nm for LF-RPE and A2E-RPE, respectively. Emission spectra for both preparations exhibit significant red-shift when the excitation wavelength is increased (p<0.0001): for 510 nm-excitation, peak emission is at 616±8 and 615±5 nm, respectively. Excitation spectra from donor eyes are broader than those of A2E: the half-maximum wavelength is 552±9 and 494±3 nm for LF-RPE and A2E-RPE, respectively. Peak excitation occurs at 464±6 and 457±0 nm, respectively. LF-RPE excitation spectra exhibit a clear inflection between 510 and 530 nm. The ratio of excitation at 530 to that at 510 nm is 0.96±0.01 (range: 0.88-0.98) for LF-RPE, significantly higher than the ratio 0.44±0.01 observed for A2E (p<0.0001). Fetal RPE that had not accumulated A2E exhibited no detectable fluorescence, whereas AF of A2E in the fetal RPE showed the characteristic A2E spectra with no detectable influence from RPE melanin. Conclusions: The red-shift in the emission spectra for longer wavelength excitation is characteristic of both LF-RPE and A2E-RPE and may reflect a distribution of fluorophore microenvironments. The broader excitation spectrum of LF-RPE and the inflection at 520 nm strongly suggest that, in addition to A2E, at least one other fluorophore is present in RPE lipofuscin. Spectral modeling predicts that this previously unidentified fluorophore should have a maximum at 500-525 nm. High spectral resolution measurements of the excitation spectrum in-vivo will define whether that spectrum also demonstrates the second fluorophore.
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