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Journal of the Optical Society of America A

Journal of the Optical Society of America A


  • Vol. 18, Iss. 6 — Jun. 1, 2001
  • pp: 1212–1230

Macular pigment density measured by autofluorescence spectrometry: comparison with reflectometry and heterochromatic flicker photometry

François C. Delori, Douglas G. Goger, Billy R. Hammond, D. Max Snodderly, and Stephen A. Burns  »View Author Affiliations

JOSA A, Vol. 18, Issue 6, pp. 1212-1230 (2001)

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We present a technique for estimating the density of the human macular pigment noninvasively that takes advantage of the autofluorescence of lipofuscin, which is normally present in the human retinal pigment epithelium. By measuring the intensity of fluorescence at 710 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, we can make accurate single-pass measurements of the macular pigment density. We used the technique to measure macular pigment density in a group of 159 subjects with normal retinal status ranging in age between 15 and 80 years. Average macular pigment density was 0.48 ± 0.16 density unit (D.U.) for a 2°-diameter test field. We show that these estimates are highly correlated with reflectometric (mean: 0.23±0.07 D.U.) and psychophysical (mean: 0.37±0.26 D.U.; obtained by heterochromatic flicker photometry) estimates of macular pigment in the same subjects, despite the fact that systematic differences in the estimated density exist between techniques. Repeat measurements over both short- and long-time intervals indicate that the autofluorescence technique is reproducible: The mean absolute difference between estimates was less than 0.05 D.U., superior to the reproducibility obtained by reflectometry and flicker photometry. To understand the systematic differences between density estimates obtained from the different methods, we analyzed the underlying assumptions of each technique. Specifically, we looked at the effect of self-screening by visual pigment, the effect of changes in optical property of the deeper retinal layers, including the role of retinal pigmented epithelium melanin, and the role of secondary fluorophores and reflectors in the anterior layers of the retina.

© 2001 Optical Society of America

OCIS Codes
(170.3890) Medical optics and biotechnology : Medical optics instrumentation
(300.0300) Spectroscopy : Spectroscopy
(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence
(330.4300) Vision, color, and visual optics : Vision system - noninvasive assessment
(330.5510) Vision, color, and visual optics : Psychophysics

Original Manuscript: May 23, 2000
Revised Manuscript: September 14, 2000
Manuscript Accepted: November 8, 2000
Published: June 1, 2001

François C. Delori, Douglas G. Goger, Billy R. Hammond, D. Max Snodderly, and Stephen A. Burns, "Macular pigment density measured by autofluorescence spectrometry: comparison with reflectometry and heterochromatic flicker photometry," J. Opt. Soc. Am. A 18, 1212-1230 (2001)

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  33. An error in DAF(460)occurs if the excitation spectrum of tissues posterior to the MP at the fovea is not proportional to that at the perifovea. We model this; we equate foveal fluorescence as FF*(Λ, λ)=kFP*(Λ, λ)+Δ(Λ, λ),where FF*and FP*are the fluorescences at the fovea and at the perifovea, respectively, k is a constant, and Δ accounts for the spectral difference between the fovea and the perifovea (can be negative). Substitution of FF*in Eq. (4) and derivation of Eq. (5), assuming that Δ/FP*≪1,gives the following approximation for the measured density: DAF′(460)≈DAF(460)+k log(e)Kmp(Λ1)-Kmp(Λ2)×Δ(Λ1, λ)FP*(Λ1, λ)-Δ(Λ2, λ)FP*(Λ2, λ).If the foveal excitation spectrum is shifted toward shorter wavelengths, then Δ decreases with increasing Λ (positive to negative), and the term in square brackets >0, resulting in an overestimation of the MP density. The opposite will occur if the shift is toward longer wavelengths.
  34. Equivalent reflectances of the fundus use a perfect Lambertian reflector located at the retina of an artificial eye as reference.21,22
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  36. For a subset of the population, we performed the fluorescence measurements at the peripheral site twice in the following order: excitations at 550, 510, 470, 430, 550, 510, and 470 nm. Rods were bleached only by the illumination and focusing lights before the first 550-nm exposure, whereas they were fully bleached (≈98%) by four excitation exposures before the second 550-nm exposure. For 30 subjects, the fluorescence at the second 550-nm exposure was 1.023±0.035times higher than that at the first exposure (p=0.001).This corresponds to a single-pass rod density of 0.02±0.03 D.U.at 500 nm. Thus approximately 60%–80% of the rods were bleached by the illumination and focusing lights (assuming a single-pass rod density of 0.05–0.1 D.U. at 500 nm).
  37. The distribution of retinal irradiance and sensitivity of the fluorometer in the 2° test field was measured optically by displacing a small reflecting surface (equivalent to ≈0.2° diameter) in a focal plane located in front of the instrument. The product of both distributions at different radii was 1.00, 1.00, 0.90, 0.80, and 0.20 at 0°, 0.25°, 0.50°, 0.75°, and 1.00°, respectively.
  38. The conversion factor used in this study was slightly larger than that calculated by using Eq. (10) from other MP spectra cited in the literature. A difference was found of 1% for the spectrum of a mixture of liposome-bound lutein and zeaxanthin in Bone et al.,4of 5% for the psychophysically determined spectrum in Wyszecki and Stiles,5and of 6% for the spectrum in fixed primate retinas obtained by microspectrometry in Snodderly et al.3
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  43. We assume that the MP distribution D(r)is exponential12,17and that it is given by D(r)=D(0)10-2 log(2)(r/FWHM),where r is the distance to the center. To convert DHFPestimated with a 0.8°-diameter field to equivalent values for a 2° test field, we further assumed that the peak density D(0)and the full width at half-maximum (FWHM) were linked by the experimentally derived relation D(0)=0.13+0.22FWHM.17 Using various values of FWHM, we obtained a nonlinear relationship between the densities for the 2°- and the 0.8°-diameter field: D2=-0.03+0.31D0.8+1.14D0.82(for D0.8<0.8 D.U.). Tp predict the densities DAFmeasured by the AF method (test field of radius R), we calculate DAF=log∑0RA(r)P(r)rΔr-log∑0RA(r)P(r)10-D(r)rΔr, where P(r)is the distribution of fundus AF in the absence of MP and A(r)is the product of the distribution of retinal irradiance and sensitivity of the fluorometer. Densities DAFwere computed with a constant P(r),the measured distribution A(r),37and an increment of r=0.005°.
  44. Most older subjects in this study and subjects in the study of Hammond et al.16were recruited from the Harvard Cooperative Program on Aging (Roslindale, Mass.), an organization devoted to good health through research and education.
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  77. Reflectances Raare positively correlated with age (r=0.4,p<0.0001) as a direct result of the increase with age of media-corrected reflectances RF′(470).This increase in reflectance cannot be accounted for by known age-related retinal changes. As we mentioned before,35we believe that this increase may result from a slight overestimation of our media correction method.
  78. The foveal fluorescence FF′in relation (21) was expressed as a function of the reflectance RF*of layers posterior to the MP [as in Eq. (6)] by RF′(λ)=Ra(λ)+RF*(λ)10-2Kmp(λ)DAF,c(460).The parameter Ra/RF*is then the single free parameter in the fits.
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  83. Direct measurements74of the ILM reflectance ρ used a perfect mirror as reference (with curvature equal to that found in the eye). To convert this reflectance into an equivalent reflectance (reference: perfect diffuser),34we used Eqs. (27) and (28) of Gorrand and Delori’s paper.74The equivalent reflectance of the ILM is then RILM=(πρr2)/(4A),where the reflectance is ρ=0041 %±0.0019 %(seven young subjects), ris the radius of curvature of the foveal depression (r=1.22±0.22 mm),and A is the area of the retinal test field (0.26 mm2for a 2°-diameter field). Using the original data (n=7, ages: 16–26 years), we found that RILM=0.019 %±0.013 % (range: 0.007%–0.04%).
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  87. F. C. Delori, D. G. Goger, B. R. Hammond, D. M. Snodderly, S. A. Burns, “Foveal lipofuscin and macular pigment,” Invest. Ophthalmol. Visual Sci. 38, S591 (1997) (ARVO Abstract No. 1657).

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