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Human macular pigment assessed by imaging fundus reflectometry
~2-6989/89 $3.00 + 0.00 Copyright Q 1989 Pergamon Press pie
Vision Res. Vol. 29, No. 6, pp. 663-674, 1989 Printed in Great Britain. All rights reserved
HUMAN
MACULAR PIGMENT ASSESSED BY IMAGING FUNDUS REFLECTOMETRY
PAUL E. K’ILBRIDE, KENNETH
R.
ALEXANDER*, MARLENE FISH&IAN
and GERALD A. FISHMAN Department
of Ophthalmology, University of Illinois at Chicago, College of Medicine, 1855 W. Taylor St, Chicago, IL 60612, U.S.A. (Receiued 28 March 1988; in revised form
I I October 1988)
Abstract-A computerized, television-based, imaging fimdus reflectometer was used to obtain estimates of the spatial distribution of macular pigment (xanthophylls) from seven normal subjects. Digitized images of the bleached macula of each subject were acquired at illuminating wavelengths from 462 to 697 nm. An analysis of spectral ceflectances indicated that differences in short-wavelength reflectance between the fovea1 center and parafovea were influenced by spatial variations in melanin and oxyhemoglobin absorption as well as by the distribution of macular pigment. To provide an estimate of the spatial distribution of macular pigment alone. we have corrected fundus images obtained at 462 nm for the effect of melanin and oxyhemoglobin absorption. The spatial variation in macular pigment double density across the horizontal and vertical meridians of the retina was well described by Gaussian functions. The peak double densities for the individual subjects ranged from 0.22 to 0.45 and the standard deviations of the Gaussian functions averaged approx. I’. Macular pigment
Xanthophyll
Melanin
Imaging
Fundus reflectometry
Cones
Fovea
has also been proposed that xanthophylls may protect the photoreceptors from light damage, The macular yellow pigment of the human both by absorbing those wavelengths that are retina is a group of short-wavelength absorbing, potentially most damaging and by scavenging nonbleaching pigments consisting primarily of the cytotoxic free radicals that are generated by zeaxanthin, which is dominant in the center of photoreceptor light absorption (Nussbaum et the fovea, and lutein, together with other minor, al., 1981; Kirschfeld, 1982). As a protective as-yet-unidentified, short-wavelength-absorbing mechanism, macular pigment may play a role in components (Snodderly, Brown, Delori & the phenotypic expression of retinal disorders, Auran, 1984a; Bone, Landrum & Tarsis, 1985; such as the development of bull’s_eye lesions in Bone, Landrum, Fernandez & Tarsis, 1988a; various maculopathies (Weiter & Delori, 1986). Bone, Landrum, Martinez & Guzman, 1988b; Since macular pigment appears to have imHandelman, Dratz, Reay & Kujik, 1988). The portance for the maintenance of normal retinal macular pigment is located primarily within the function, it is of interest to develop a nonreceptor axon and inner plexiform layers of the invasive procedure to measure spatial variations macula (Snodderly et al., 1984a). The spatial in macular pigment in the intact human eye. distribution of the macular pigment corre- Previous reports (Brindley & Willmer, 1952; sponds approximately to that of the cone photo- Ishak, Housni, Said & Abd El-Sayed, 1967; Van receptors; the pigment is most dense within the Norren & Tiemeijer, 1986) have suggested that foveola and decreases to a low constant Ievel by analysis of fundus reflectance may provide such approx. 4“ of eccentricity (Snodderly et al., a measure of macular pigment distribution. In 1984a; Snodderly, Auran & Delori, 1984b; those studies, the short-wavelength reflectance Bone et al,, 1988a). of the bleached fovea was compared to that of It has been suggested that, by absorbing a region of the bleached parafovea in order to short-wavelength light, the macular pigment derive the “two-way” or “double” density [S(i_)J may serve to reduce the effects of chromatic of macular pigment: aberration and light scatter on visual perfl> formance (Nussbaum, Pruett & Delori, 198 1). It ___where R,(A) and R,(J) represent the measured *To whom reprint requests should be addressed. INTRODUCTION
663
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PACL E. KILBRIDE et al
reflectances of the parafovea and fovea, respectively. It should be noted that zj(E.) is influenced by light scatter, which is light returned to the measuring instrument that has not passed twice through the pigment-containing layers of the retina (cf. King-Smith. 1973). Consequently, 6(j.) is not equivalent to twice the optical density of macular pigment. In previous reflectometric studies (Brindley & Willmer, 1952; Ishak et al., 1967; Van Norren & Tiemeijer, 1986), reflectances were measured only at discrete, widely separated retinal loci, so that estimates of the spatial distribution of macular pigment were not provided. In addition, the possibility that other short-wavelength absorbing pigments besides macular pigment might influence reflectance differences between the fovea and parafovea was not considered quantitatively. As discussed by Van Norren and Tiemeijer (1986), four ocular pigments (macular pigment, lens pigment, melanin, and oxygenated hemoglobin) are the primary determinants of the reflectance of the bleached fundus. Melanin has a comparatively high short-wavelength absorption (Hunold & Malessa, 1974) and a peak optical density within the fovea1 region of the pigment epithelium and choroid (Weiter, Delori, Wing & Fitch, 1986). Hemoglobin also has a comparatively high short-wavelength absorption (Mook, Assendelft & Zijlstra, 1969), and a relatively greater absorption in the parafovea than in the fovea1 avascular zone. In a previous study, we provided evidence that spatial variations in melanin and oxyhemoglobin absorption have influenced earlier reflectometric measurements of macular pigment (Alexander, Kilbride, Fishman & Fishman, 1987). In the present report, we describe a method for assessing the spatial distribution of macular pigment in normal subjects, using a television-based, imaging fundus reflectometer, in which the presumed effects of melanin and oxyhemoglobin are minimized. In this procedure, a digitized fundus image obtained at an illuminating wavelength of 559 nm (governed primarily by melanin and oxyhemoglobin absorption) is subtracted from a digitized image obtained at 462 nm (determined by macular pigment absorption as well as by melanin and oxyhemoglobin) to obtain the spatial variation of the double density of macular pigment. The 559 nm image is corrected for the change in melanin and oxyhemoglobin density between 559 and 462 nm. We have applied this analysis to seven normal individuals to deter-
mine the degree of intersubject variability in the double density of macular pigment among this group. METHOD
Subjects
Seven individuals with normal vision participated in the study. All were Caucasian with a best-corrected Snellen visual acuity of 20120 or better and normal color vision as assessed with a Nagei anomaloscope. The mean age of the subjects was 33 yr; the range was 22-39 yr (Table I). Older subjects were excluded to minimize the possibility of lens scatter (Sigelman, Trokel & Spector, 1974). Informed consent was obtained from all subjects after the nature of the procedure had been explained fully. Apparatus
The television-based fundus reflectometer used to measure fundus reflectance has been described previously (Kilbride. Read, Fishman & Fishman, 1983). Briefly, a Sierra Scientific video camera containing an RCA Type 4804 silicon intensifier target (SIT) camera tube viewed the fundus through the optics of a modified Zeiss fundus camera. A fiberoptic light guide illuminated by a computer-controlled optical system replaced the normal Zeiss light source. In this optical system, light from a 150-watt xenon arc lamp passed successively through an infrared blocking filter; a neutral density wedge and an interference fiber wheel, both of which were driven by stepping motors; and an electromechanical shutter. The spectral characteristics of the measurement light were defined by 13 interference filters with peak wavelengths that ranged from 462 to 697 nm in approx. 20 nm steps and half-height bandwidths of approx. 10 nm. The output of the video camera was digitized by a high-speed analog-todigital converter, with an image resolution of 112 by 100 pixels (each pixel subtended 0.1” of visual angle) and 256 levels of gray scale resolution. The digitized images were stored on a magnetic disk and were later transferred to magnetic tape for archival storage. Procedure
The procedure used to acquire the fundus images was identical to that described previously for studies of cone pigment density differences (Kilbride et al., 1983). TO provide calibrations for the fundus reflectance of the
665
Image analysis of macular pigment Table 1. Subject ages, parameters of individual macular pigment distributions
(1) Subject No.
(2)
Mean SD
is)
(7) e(o) Vertical
(8) r.m.s. Error Horizontal
(9) r.m.s. Error Vertical
Sex
Age
M
22 30 31 36 36 37 39
0.37 0.35 0.33 0.22 0.26 0.30 0.44
0.40 0.36 0.33 0.27 0.29 0.35 0.45
0.85 1.10 I .03 1.06 0.78 0.87 1.37
0.87 0.89 0.92 0.98 0.81 0.93 1.17
0.03 0.03 0.02 0.03 0.02 0.03 0.02
0.04 0.03 0.03 0.03 0.03 0.04 0.03
33 5.8
0.32 0.07
0.35 0.06
1.01 0.20
0.94 0.12
0.03 0.01
0.03 0.01
a$, Vertical
eye, an artificial eye with a focal length of 24 mm and with an imaging surface of Halon powder was scanned prior to subject testing. The spatial distribution of light in the digitized image of the artificial eye was used to correct the digitized fundus images for spatial nonuniformities in the radiometric response of the imaging system, while the reflectance values of the artificial eye allowed comparisons of fundus reflectances among subjects. The pupil of the test eye was dilated and accommodation paralyzed with 2.5% phenylephrine hydrochloride and 1% cyclopentolate hydrochloride drops. Head position during testing was maintained by a dental impression bite block and forehead rests; the nontested eye fixated upon a dimly illuminated target. The eye was exposed for 30 set to a 605 nm bleaching light of 6.12 log td, which is sufficient to bleach more than 97% of the middle- and long-wavelength cone pigments (Alpern, Maaseidvaag & Ohba, 1971). Immediately after the offset of the bleaching light, a series of digitized images of the bleached fundus was obtained with illumination provided by monochromatic light from each of the 13 interference filters. The exposure duration of each wavelength was 83 msec (5 video fields), during which time a fundus image was electronically integrated on the camera tube. The interval between exposures was approx. 220 msec, and each complete wavelength series was obtained in about 4 sec. In the present study, either two (for 2 subjects) or three (for 5 subjects) complete wavelength series were obtained, with each series preceded by a bleaching exposure. The computer adjusted the light radiance at each wavelength so that the camera operated near the center of its linear range. This procedure was necessary since the output of the video camera is linear only within an input range of 10 : 1, while fundus spectral reflectance varies by more than 100: 1. The measurement human
(6)
(4) 6’ Hori.%rtal
F M F M F M
(3)
a(o) Horizontal
lights produced minimal bleaching of the middle- and long-wavelength cone photopigments and were considerably below the maximum permissible exposure (MPE) for retinal irradiance. The highest retinal illuminance was 4.57 log td-set (at 559 nm), which would bleach approx. 0.7% of cone photopigments in the dark-adapted eye (Alpern et al., 1971). The highest retinal irradiance was 2.47 x 10m4W cme2 (at 462nm), which is more than 4 log units below the MPE at our exposure duration (Delori, Parker & Mainster, 1980). For each subject, each of the digitized images of the fundus was aligned with a selected anchor image to compensate for any small eye movements that may have occurred between the periods of fundus illumination. To accomplish the image registration, the fundus image at a given wavelength was alternated rapidly with the anchor image on a video monitor. Using a trackball system, the operator moved the video image of the fundus until apparent movement of retinal detail was minimized. As a result of this image alignment, the reflectance factors of corresponding retinal regions could be compared across wavelengths. For each subject, the multiple fundus images obtained at each wavelength were averaged to provide a mean digitized fundus image for that subject at that wavelength. In the following image analysis, as in previous studies (Brindley & Willmer, 1952; Van Norren & Tiemeijer, 1986), reflectance factors were obtained. The reflectance factor [R,, (i.)] of each pixel (coordinates x and y) represents the ratio of the radiant intensity of that pixel in the digitized image of a human fundus [Z,l,Y,,.j,] to the radiant intensity of the corresponding pixel in the digitized image of the artificial eye [ZC,,Y,,,j,]:
(2)
PAUL E. KILBRIDE et al
666 RESULTS
An individual series of digitized images of the bleached fundus of a typical normal subject, obtained with monochromatic illumination, is presented in Fig. 1. At short wavelengths of illumination (top row) there is a region of low reflectance within the fovea that is not apparent at longer wavelengths (bottom row). The spatial and spectral characteristics of this region are suggestive of macular pigment. However. we have reported previously that macular pigment is not the sole determinant of this shortwavelength reflectance pattern (Alexander et al., 1987). The following analysis indicates the validity of this proposal for the present group of normal subjects. Our analysis is similar to that of Van Norren and Tiemeijer (1986) in which macular pigment, lens pigment, melanin, and oxygenated hemoglobin are considered to be the primary determinants of the reflectance of the bleached fundus. All of these pigments have some degree of absorption at short wavelengths. In accordance with Van Norren and Tiemeijer (1986) we assume that these pigments obey Beer’s law and are located in front of a spectrally neutral reflector (sclera), the properties of which are the same between the fovea and parafovea. Figure 2 presents the density spectra of these pigments, which, except for oxyhemoglobin, are plotted directly from published values. The density spectra for macular pigment and the lens were obtained from Wyszecki and Stiles (1982) while that for melanin is replotted from Hunold and Malessa (1974). The density spectrum for oxyhemoglobin is 0.02 times the values of Mook et al. (1969) which approximates the optical
Wavelength
3 Wavelength
(nm)
Fig. 4. Mean log spectral reflectances of the fovea and parafovea of the seven normal subjects. Error bars indicate ) 1SEM.
density of oxyhemoglobin in the parafovea of the human fundus (cf. Van Norren & Tiemeijer, 1986). To demonstrate the contribution of these ocular pigments to reflectance differences between the fovea and parafovea, we first determined the average spectral reflectance of two windows within the fundus image, as depicted in Fig. 3. The fundus image in this figure was obtained at 462 nm, and is identical to that illustrated in Fig. 1. The central window, indicated by the circle, is 0.7” in diameter. The outer window, indicated by the ring, has an inner diameter of 6” and an outer diameter of 7 I. This latter value was the limit of the common area for the 244 fundus images obtained from the seven subjects. The mean log spectral reflectances of these two windows are shown in Fig. 4. In agreement with previous studies (Brindley & Willmer, 1952; Van Norren & Tiemeijer, 1986; Kilbride, Fishman, Hutman & Fishman, 1986), the log reflectances of both the fovea and parafovea are lowest at short wavelengths and increase with increasing wavelength. Moreover, there is a systematic difference between the mean log spectral reflectances of the fovea and parafovea, as reported previously (Brindley 8t Willmer. 1952; Van Norren & Tiemeijer, 1986) with the greatest difference at short wavelengths. We have plotted this difference [p(k)], which is equivalent to the log ratio of parafoveal to fovea1 reflectance factors, in Fig. 5:
(run)
Fig. 2. Density spectra of macular pigment, lens pigment, melanin, and oxyhemoglobin.
(3
Fig. 1. Digitized fundus images of a typical normal subject obtained with the following wavelengths of illumination (left to right): 462, 480, 502, 521 nm (top); 541, 559, 580, 598 nm (middle); 617, 634, 657, 675 nm (bottom). An additional image, obtained at 697 nm and essentially identical to that at 675 nm, was omitted for lack of space. (Due to limitations inherent in the reproduction process, these photographs do not accurately represent the actual digitized fundus images.)
Fig. 3. Digitized fundus image obtained at 463 nm of illumination (from Fig. I). The circle and ring illustrate the retinal regions whose pixel values were averaged to obtain estimates of fovea1 and parafoveai spectral reflectance, respectively. 667
669
Image analysis of macular pigment
---
0.5
0.4
_____.
~
r.
500
-
-._
-
550 Wavelength
MaMCulllrPioment Melanin “WlO~bbt”
camblned
600
650
700
(nm)
Fig. 5. Mean log reflectance ratios @(A)] between the parafovea and fovea. The solid line is a least-squares fit of the combined density spectra of macular pigment, melanin, and oxyhemoglobin. The interrupted lines show the constituent density spectra of the individual ocular pigments. Error bars indicate If: 1SEM.
Since the greatest values of p(I) occur at short wavelengths, previous studies (Brindley & Willmer, 1952; Van Norren & Tiemeijer, 1986) have presumed that p (A) is due only to macular pigment absorption. However, the presence of nonzero values of p (A) at long wavelengths, also seen in previous studies (Brindley & Willmer, 1952; Van Norren & Tiemeijer, 1986), suggests that macular pigment is not the only contributor. To illustrate the relative contribution of ocular pigments besides macular pigment to the p(A) values, we used a least-squares procedure to fit the data in Fig. 5 by the combined density spectra of macular pigment, melanin, and oxyhemoglobin:
where Di(n) represents the density spectrum of melanin, macular pigment, or hemoglobin, respectively, and a, are derived weighting coefficients. In this analysis, we assume that lens absorption is the same for both retinal regions, since the measurement light passes through the crystalline lens in a focused beam. The solid line in Fig. 5 represents the best-fit combined density spectrum. The squared multiple regression coefficient (mr2) was 0.98; the standard error of the estimate was 0.04. The interrupted lines represent the values of aiDi for the individual pigments. At wavelengths longer than approx. 530 nm, melanin and oxyhemoglobin appear to be the major determinants of p(l). At short wavelengths, p (J.) is apparently determined
jointly by macular pigment and melanin, with a minor contribution from oxyhemoglobin. Therefore, spatial variations in macular reflectance at short wavelengths do not represent the distribution of macular pigment alone. The following analysis describes a method for reducing the effects of melanin and oxyhemoglobin absorption on reflectometric estimates of macular pigment double density. We first apply this analysis to the mean data of the seven subjects, and then present results from individual subjects. Figure 6a plots the log reciprocal reflectance factor (log [ 1/R.,,:,,,]) of each pixel in the mean image of the seven subjects, obtained at 462 nm. To reduce pixel noise, the mean values in this figure and in Fig. 6b were linearly filtered by a moving average filter, in which each pixel is replaced by an average of its neighbors (a 3-by-3 array with equal weight for each pixel). As noted above, the spatial pattern of reflectance at 462 nm is not due entirely to macular pigment; melanin and oxyhemoglobin also contribute. Therefore, to determine the spatial variation in macular pigment alone, it is necessary to remove the influence of melanin and oxyhemoglobin from the data in Fig. 6a. This can be accomplished by subtracting a fundus reflectance distribution obtained at a wavelength that represents predominantly melanin and oxyhemoglobin absorption. It is evident from the data in Fig. 5 that wavelengths longer than 530 nm can provide such a distribution. It is also necessary that the change in the extinction coefficient between 462 nm and the chosen wavelength be equivalent for both melanin and oxyhemoglobin. The reflectance data at a wavelength of 559 nm fulfill these two criteria. The mean log reciprocal reflectance factor of each pixel at 559 nm (log ( I/Rr.r.559])is presented in Fig. 6b. This surface was multiplied by the average change in the extinction coefficients of melanin and oxyhemoglobin (1.14) that occurs between 559 and 462 nm. Finally, the product was subtracted from the data of Fig. 6a to derive the corrected double density [6&J of macular pigment:
- 1.14*loisWRx,y,d
(5)
It should be noted that lens density was not included in this analysis. Absorption by the lens adds a uniform constant to each reflectance
670
PAUL E. KILBRIDEet al.
surface but does not affect the shape of the surface. Moreover, the change Ott lens density between 462 and 559 nm, for the age range of our subjects, is not of sufficient magnitude to alter the results significantly- iwyszecki and Stiles. 1982). The result of thus image subtraction is shown in Fig. 6~. According to our analysis, this surface represents the spatial variation in the double density of macular pigment across the central fovea. To examine the symmetry toi the surface shown in Fig. 6c, we have plotted isodensity contours in Fig. 7. The spatial variation of a;,; is approximately symmetrical. To illustrate further the spatial distribution of ilk) and to indicate the degree of intersubject variability, we have plotted cross-sections of Fig. 6c, measured along the horizontal and vertical meridians, in Fig. 8a and b, respectively. To obtain these cross-sections, the image analysis described above (equation 5) was applied separately to each individual subject, and values of S& for each subject were obtained along the horizontal and vertical meridians. Each point in a crosssection of an individual subject represents an average of a region 3 pixels wide centered on that point and orthogonal to the measured meridian. The hatched regions in Fig. 8a and b represent the mean cross-sections of the seven subjects + I SEM, measured along the horizontal and vertical meridians, respectively. The solid lines in Fig. 8 represent Gaussian functions of the form:
Fig. 6. Mean log reciprocal of fundus reflectance at 462 nm (a); 559 nm (b): and 462 nm after correction for absorption by melanin and oxyhemoglobin (c). See text for details.
where u and h are scaling parameters, x indicates eccentricity, and 1~and D are the mean and standard deviation of the dtstribution, respectively. This equation was tit to the mean data for each of the two meridians by an iterative curve-fitting program with a leastsquares criterion. Values of p and (T for the mean data were 0.13 and 0.99’ for the horizontal meridian, and 0.09 and 0.94 for the vertical meridian. For both meridians, the r.m.s. error was 0.01. As a second measure of individual variation, Gaussian functions were also ht separately to the horizontal and vertical meridians for each of the individual subjects. An estimate of a&,, at the fovea1 center for each subject for each meridian was obtained from the difference between the value of 0 & at the peak of the best-fit Gaussian function and the value of ~3;~~ at the asymptotic
Image analysis of macular pigment
4-3~ .,,,,, ~
),,,(,
-3
-2
-1
0
1
2
3
Degrees
Tem#mnl
1
HamI
Fig. 7. Isodensity contours of the surface shown in Fig, 6c.
level. These values of 6;,, for the horizontal and vertical meridians are given in Table 1 (columns 4 and 5, respectively). Values of c for the individual subjects, representing the spatial variation in S&, along the horizontal and vertical meridians, are also presented in Table I (columns 6 and 7, respectively). The r.m.s. errors of the best-fit Gaussians are given in Table I (columns 8 and 9, respectively).
Discussion
The spatial variation in the double density d&s of macular pigment that we have derived from imaging fundus reftectometry (Figs 6-8) is similar to previous measures obtained within the human retina by psychophysical methods (Stabell & Stabell, 1980; Pease & Adams, 1983; Vienot, 1983; Moreland & Bhatt, 1984;
671
Werner, Donnelly & Kliegel, 1987) and within the monkey retina by microdensitometry (Snodderly et al., 1984b). In those studies, the macular pigment absorbance was highest in the fovea1 center and declined to a low constant level by approximately 4” of eccentricity. In addition to estimates of macular pigment absorption, our data also provide an indication of spatial variations in melanin absorption within the macula. Melanin is a screening pigment within the retinal pigment epithelium and choroid that may have a protective antioxidant function (Ostrovsky, Sakina & Dontsov, 1987). A reduction in melanin, for example, has been implicated in the development of retinal disorders such as age-related macular degeneration (Weiter et al., 1985). Within the choroid, the density of melanin is highest in the fovea1 center (Weiter et al., 1986). Within the pigment epithelium, melanin density tends to be higher in the far periphery than in the macular region (Schmidt & Peisch, 1986), but there is an absorption peak within the central fovea (Weiter et al., 1986). As a consequence, the combined melanin density of the pigment epithelium and choroid is greatest in the fovea1 center. Our measurements are consistent with these previous reports. The reflectance data at 559 nm, shown in Fig. 6b, represent prima~ly the spatial variation in melanin absorption. There is a peak absorption in the fovea1 center, in agreement with opticai measurements in autopsy eyes (Weiter et al., 1986). Our estimates of the double density of macular pigment within the central fovea are lower than values previously obtained by fundus reflectometry (Brindley & Willmer, 19.52; 0.4
tbl
3 g
0.3
0 e d t
0.2
8 _I
11 -3.0
s
1
s
1
-2.0 -1.0
iemporai
‘
13 0.0
11
1.0
Eccentricity(Degf
I
2.0
*
4
3.0 NPIdl
Eccentricity (De&
Superior
Fig. 8. The spatial pattern of macular pigment absorption at 462 nm across the horizontal (a) and vertical (b) meridians of the visual field. The hatched regions show means i: i SEM. The solid lines represent Gaussian functions fit by a least-squares procedure to each of the two meridians.
672
PAUL E. KILBRIDEet al
Van Norren & Tiemeijer, 1986). This is due primarily to the fact that we have attempted to eliminate the contribution of melanin and oxyhemoglobin in our analysis. In previous studies (Brindley & Willmer, 1952; Ishak et al., 1967; Van Norren & Tiemeijer, 1986), values of p(%), representing the difference between the Iog reflectances of the parafovea and fovea, were used as a measure of macular pigment density. However, our results indicate that shortwavelength reflectances should be corrected for melanin and oxyhemoglobin absorption before accurate estimates of macular pigment double density can be derived. Since our values of p (i.) are comparable to those of earlier reports (Brindley & Willmer, 1952; Van Norren & Tiemeijer, 1986), our estimates of S;,, are necessarily lower than in those studies. Our macular pigment double densities are also lower than might be expected from psychophysical studies, in which optical densities average approx. 0.5 (e.g. Werner et al., 1987; Wyszecki & Stiles, 1982). Since there appears to be considerable intersubject variability in psychophysi~lly measured macular pigment density (Werner et al., 1987; Pease, Adams & Nuccio, 1987). it is possible that our subjects represent the lower range of the distribution. However, it is more likely that the apparent discrepancy results from fundamental differences between psychophysical and reflectometric methods. One of the primary factors is light scatter, which is an unavoidable influence on reflectometric estimates of the double density of macular pigment, as well as of photoreceptor photopigment double density (Rushton, 1965; King-Smith, 1973; Margolis, Siegel & Ripps, 1987). While a fraction of the light reaching the measuring instrument has passed twice through the layer of macular pigment, an unknown proportion has passed through only once, or not at all. Some of the light may have been scattered within the retina or reflected specularly from the internal limiting membrane, particularly in the eyes of younger subjects (Delori, Gragoudas, Francisco & Pruett, 1977). In addition, light from the measuring beam may have been back-scattered from the cornea and/or lens or forward-scattered in its return through the lens center (Sigelman et al., 1974). All of these factors would tend to reduce the contrast of the fundus images and could therefore result in a lower estimate of macular pigment absorption than would be expected from psychophysical studies. Moreover, refiectometric estimates of
macular pigment spatial distribution may be inff uenced by the optical properties of the fovea1 pit (Williams, 1980). While light scatter poses a problem for reflectometric estimates of the spatial distribution of macular pigment, the use of psychophysical procedures to assess spatial variations in macular pigment atso has potential limitations. Psychophysical studies measure the screening effect of macular pigment on light that has been absorbed by cone photoreceptors. As a consequence of the Stiles&?rawford effect. light that is of normal incidence tfn the photoreceptors is more likely to be absorbed than light that is scattered within the retina (Crawford, 1972). Since macular pigment is primarily contained within the inner retimi (Snodderly et al., 1984a), the psychophysically measured optical density of macular pigment represents primarily the density of pigment interposed between the individual photoreceptors and the exit pupil of the eye. However, evidence indicates that the psychophysically measured density of macular pigment represents the density at the edge of a test flash. rather than an average of the stimulated region (Werner et al., 1987). Consequently, there could be a considerable reduction in the density of macular pigment within specific regions of the fovea without necessarily a decrease in the psychophysically measured optical density of macular pigment. The optimum procedure for the psychophysical measurement of the spatial distribution of macular pigment would be the presentation of a tiny test flash at a series of retinal locations (e.g. Williams, MacLeod & Hayhoe, 1981), a timeconsuming procedure requiring precise fixation. Consequently. despite the limitations of light scatter. our method for assessing macular pigment by imaging fundus reflectometry can potentially provide information about spatial patterns of macular pigment within the intact human retina, both normal and abnormal, that is not obtained easily by alternative methods. Moreover, the method has potential application to the study of macular pigment distribution in animal retinas as well. Rcknowfedgements--Theauthors thank Morton F. Goldberg and Neal S. Peachey for helpful comments on the manuscript, Robert J. Anderson for statistical advice, and Kathleen Louden for editorial assistance. The research was supported by NE1 Grants EY04848, EYO6589, Core Grant EY01792, and a center grant from the National Retinitis Pigmentosa Foundation Fighting Blindness, Baltimore. MD.
Image analysis of macular pigment REFERENCES
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expressivity in fundus albipunctatus. Ophthaimalagy. 94, 141G1422.
Alexander, K. R.. Kilbride, P. E., Fishman, M. & Fishman, G. A. (1987). Macular pigment and reduced fovea] short-wavelength sensitivity in retinitis pigmentOSa. Vision Research, -77. 1077-l 083. ,bJpern,M., Maaseidvaag, F. & Ohba, N. ($971). The kinetics of cone visual pig!IIentS in man. f’?km hW2ar& I I, 539-549. Bone, R. A., Landrum, J. T. & Tarsis, S. L. (1985). Preliminary identification of the human macular pigment. Vision Reseurch, 25‘ 1531-153s. Bone. R. A., Landrum. J. T., Fernandez, L. & Tarsis, S. L. (1988a). Analysis of the macular pigment by HPLC: Retinal distribution and age study. Inuesrigurive Ophthutmology and Visual Science, 29, 843-849.
Bone, R. A., Landrum, J. T., Martinez, J. L. & Guzman, S. M. (1988b). In vitro vs in uivo optical density of the macular pigment. Inveszigafil~eOp~lhatmatogy and Visual Science, 29 (SuppI.), 446. Brindiey, G. S. & Willmer, E. N. (1952). The retlexion of light from the macular and peripheral fundus oculi in man. Journal of Physiology, London, 116, 350-356. Crawford, B. H. (1972). The Stiles-Crawford effects and their significance in vision. In Jameson, D. & Hurvich, L. M. (Eds.) ~andboak o/sensory physiaiogy W/4, visual psychophysics (pp. 470-483). Berlin: Springer. Delori, F. C., Gragoudas, E. S., Francisco, R. & Pruett, R. C. (1977). Monochromatic ophthalmoscopy and fundus photography. The normal fundus. Archives of Ophrhalmoiog~~. 95. 861-868.
Delori. F. C., Parker, J. S. & Mainster, M. A. (1980). Light levels in fundus photo~aphy and fluorescein angiography. Vision Research, 20, 1099-1104. Handelman G. J.. Dratz, E. A., Reay, C. C. & Kujik, F. J. G. M. Van (1988). Carotenoids in the human macula and whole retina. Imesrigutive Ophrhatmotogy and Visual Science. 29. 850-855.
Hunold. W. & Maiessa. P. (1974). Spectrophotometric determination of the melanin pi~entation of the human ocular fundus in riw. Oph~~almotogy Research, 6, 355-362.
Ishak. 1. G. H.. Housni, F. A., Said, F. S. & Abd El-Sayed, S. Z. (1967). Measurement of the absorption spectrum curve of the yellow macular pigmentation in the living eye. In Weigelin. E., Wittels, L. & Francois, .J. (Eds.) XX cancilium op~~halmatagi~um, acta, Munich, 1966 (pp. 1101-t 108). Excerpta Medica, Amsterdam. Kilbride, P. E., Read, J. S., Fishman G. A. & Fishman, M. (1983). Determination of human cone pigment density difference spectra in spatially resolved regions of the fovea. Vision Research. 23. 1341-I 350. Kilbride. P. E., Fishman. M., Hutman, L. P. & Fishman G. A. (1986). Fovea1 cone pigment density difference and reflectance in retinitis pigmentosa. Arehives qj’ Ophrhatmoiogy, 104, 220-224.
King-Smith, P. E. (1973). The optical density of erythrolabe determined by retinal densitometry using the selfscreening method. Journal of Physiology. London, 230, 535-549.
Kir~hfeld, K. (1982). Carotenoid pigments: Their possible role in protecting against photooxidation in eyes and photoreceptor cells. Proceedings of the Royal Society of London B, 216, 71-8.5.
Margolis, S.. Siegel. I. M. & Ripps, H. (1987). Variable
Mook, G. A., Assendelft, 0. W. Van & Zijlstra. W. G. (1969). Wavelength dependency of the spectrophotometric determination of blood oxygen saturation. Ctinica Chimica Acta, 26, 170-173.
Moreland, J. D. & Bhatt, P. (1984). Retina1 distribution of macular pigment. In Verriest, G. (Ed.) CO~OW ~i.Mrz deJiciencies VII (pp. 127-132). Junk, The Hague. Nussbaum, J. J., Pruett R. C. % Delori, F. C. (1981). Macular yellow pigment. The first 200 years. Rerina, 1, 29&3 IO. Ostrovsky, M. A., Sakina, N. L. & Dontsov, A. E. (1987). An antioxidative role of ocular screening pi~ents. vision Research, 27, 893-899.
Pease, P. L. & Adams, A. J. (1983). Macular pigment difference spectrum from sensitivity measures of a single cone mechanism. American Journal of Opromerry and Physiological Optics, 60, 667672.
Pease, P. L., Adams, A. J. & Nuccio, E. (1987). Optical density of human macular pigment. Vision Re.~earch, 2% 705-710.
Rushton, W. A. H. (1965). Stray light and the measurement of mixed pigments in the retina. Journal qf Physiotogy, London, t76. 46-55.
Schmidt, S. Y. & Peisch, R. D. (1986). Melanin concentration in normal human retinal pigment epithelium. Regional variation and age-related reduction. Znuestigarive Ophrhatmology and Visual Science, 27, 1063-1067. Sigelman, J., Trokel, S. L. &Spector, A. (1974). Quantitative biomicroscopy of lens light back scatter. Changes in aging and opacification. Archives qf Ophrhatmotogy, 92, 437-442.
Snodderiy, D. M., Brown, P. K., Delori, F. C. & Auran, J. D. (1984a). The macular pigment. I. Absorbance spectra, localization, and discrimination from other yellow pigments in primate retinas. Inuesligative Ophlhatmotogy and Visual Science, 25, 660-673. Snodderly, D. M., Auran, J. D. & Delori, F. C. (1984b). The macular pigment. II. Spatial distribution in primate retinas. Inves~igafive ~ph~haimaiogy and Visuaf Science, 25, 674-685.
Stabell, U. & Stabell, B. (1980). Variation in density of macular pigmentation and in short-wave cone sensitivity with eccentricity. Journal of’ the Optical Society of America, 70, 706-7 11. Van Norren, D. & Tiemeijer, L. F. (1986). Spectral refiectance of the human eye. Vision Research, 26, 313-320.
Vienbt, F. (1983). Can variation in macular pigment account for the variation of colour matches with retina1 position? In Mollon, J. D. & Sharpe, L. T. (Eds.) Colour vision physiology andpsychophysics. (pp. 107-I 16). New York, Academic Press. Weiter. J. J. & Delori, F. C. (1986). An explanation for the “bull’s eye” macular lesion. Investigative Ophrhatmatagy and Visuat Science, 27, (Suppl.), 336. Weiter, J. J.. Delori, F. C., Wing, G. L. & Fitch, K. A. (1985). Relationship of senile macular degeneration to ocular pigmentation. American JournalqfOphthatmatogy, 99, 185-187.
Weiter. J. J.. Deiori, F. C., Wing, G. L. & Fitch, K. A, (1986). Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in human eyes. Inoesrigarive OphIhatmotogy and Visual Science, 27. 145-152.
Werner. J. S.. Donnelly, S. K. & Kliegel, R. (1987). Aging
PAUL
E.
KILBRIDE
and human macular pigment density. Appended with translations from the work of Max Schultze and Ewald Hering. Vision Research, 27, 257-268. Williams, D. R. (1980). Visual consequences of the fovea1 pit. Invesligative Ophlhalmolog~~ and Visual Sciemr, 19. 653667.
et al.
Williams, D. R.. MacLeod, D. I. A. Xr Mayhoe. M. M ( 1981). Punctate sensitivity of the blue-sensitive mechanism. F’i~sion~~,.~~ff~e~, 21. 1357~I37q Wyszecki, G. & Stiles, W. S. (1982). CO~OZC sc’~encr:co~zcr~rs war/ me/hods (2nd edn). New York. Wile).
Vision Res. Vol. 29, No. 6, pp. 663-674, 1989 Printed in Great Britain. All rights reserved
HUMAN
MACULAR PIGMENT ASSESSED BY IMAGING FUNDUS REFLECTOMETRY
PAUL E. K’ILBRIDE, KENNETH
R.
ALEXANDER*, MARLENE FISH&IAN
and GERALD A. FISHMAN Department
of Ophthalmology, University of Illinois at Chicago, College of Medicine, 1855 W. Taylor St, Chicago, IL 60612, U.S.A. (Receiued 28 March 1988; in revised form
I I October 1988)
Abstract-A computerized, television-based, imaging fimdus reflectometer was used to obtain estimates of the spatial distribution of macular pigment (xanthophylls) from seven normal subjects. Digitized images of the bleached macula of each subject were acquired at illuminating wavelengths from 462 to 697 nm. An analysis of spectral ceflectances indicated that differences in short-wavelength reflectance between the fovea1 center and parafovea were influenced by spatial variations in melanin and oxyhemoglobin absorption as well as by the distribution of macular pigment. To provide an estimate of the spatial distribution of macular pigment alone. we have corrected fundus images obtained at 462 nm for the effect of melanin and oxyhemoglobin absorption. The spatial variation in macular pigment double density across the horizontal and vertical meridians of the retina was well described by Gaussian functions. The peak double densities for the individual subjects ranged from 0.22 to 0.45 and the standard deviations of the Gaussian functions averaged approx. I’. Macular pigment
Xanthophyll
Melanin
Imaging
Fundus reflectometry
Cones
Fovea
has also been proposed that xanthophylls may protect the photoreceptors from light damage, The macular yellow pigment of the human both by absorbing those wavelengths that are retina is a group of short-wavelength absorbing, potentially most damaging and by scavenging nonbleaching pigments consisting primarily of the cytotoxic free radicals that are generated by zeaxanthin, which is dominant in the center of photoreceptor light absorption (Nussbaum et the fovea, and lutein, together with other minor, al., 1981; Kirschfeld, 1982). As a protective as-yet-unidentified, short-wavelength-absorbing mechanism, macular pigment may play a role in components (Snodderly, Brown, Delori & the phenotypic expression of retinal disorders, Auran, 1984a; Bone, Landrum & Tarsis, 1985; such as the development of bull’s_eye lesions in Bone, Landrum, Fernandez & Tarsis, 1988a; various maculopathies (Weiter & Delori, 1986). Bone, Landrum, Martinez & Guzman, 1988b; Since macular pigment appears to have imHandelman, Dratz, Reay & Kujik, 1988). The portance for the maintenance of normal retinal macular pigment is located primarily within the function, it is of interest to develop a nonreceptor axon and inner plexiform layers of the invasive procedure to measure spatial variations macula (Snodderly et al., 1984a). The spatial in macular pigment in the intact human eye. distribution of the macular pigment corre- Previous reports (Brindley & Willmer, 1952; sponds approximately to that of the cone photo- Ishak, Housni, Said & Abd El-Sayed, 1967; Van receptors; the pigment is most dense within the Norren & Tiemeijer, 1986) have suggested that foveola and decreases to a low constant Ievel by analysis of fundus reflectance may provide such approx. 4“ of eccentricity (Snodderly et al., a measure of macular pigment distribution. In 1984a; Snodderly, Auran & Delori, 1984b; those studies, the short-wavelength reflectance Bone et al,, 1988a). of the bleached fovea was compared to that of It has been suggested that, by absorbing a region of the bleached parafovea in order to short-wavelength light, the macular pigment derive the “two-way” or “double” density [S(i_)J may serve to reduce the effects of chromatic of macular pigment: aberration and light scatter on visual perfl> formance (Nussbaum, Pruett & Delori, 198 1). It ___where R,(A) and R,(J) represent the measured *To whom reprint requests should be addressed. INTRODUCTION
663
664
PACL E. KILBRIDE et al
reflectances of the parafovea and fovea, respectively. It should be noted that zj(E.) is influenced by light scatter, which is light returned to the measuring instrument that has not passed twice through the pigment-containing layers of the retina (cf. King-Smith. 1973). Consequently, 6(j.) is not equivalent to twice the optical density of macular pigment. In previous reflectometric studies (Brindley & Willmer, 1952; Ishak et al., 1967; Van Norren & Tiemeijer, 1986), reflectances were measured only at discrete, widely separated retinal loci, so that estimates of the spatial distribution of macular pigment were not provided. In addition, the possibility that other short-wavelength absorbing pigments besides macular pigment might influence reflectance differences between the fovea and parafovea was not considered quantitatively. As discussed by Van Norren and Tiemeijer (1986), four ocular pigments (macular pigment, lens pigment, melanin, and oxygenated hemoglobin) are the primary determinants of the reflectance of the bleached fundus. Melanin has a comparatively high short-wavelength absorption (Hunold & Malessa, 1974) and a peak optical density within the fovea1 region of the pigment epithelium and choroid (Weiter, Delori, Wing & Fitch, 1986). Hemoglobin also has a comparatively high short-wavelength absorption (Mook, Assendelft & Zijlstra, 1969), and a relatively greater absorption in the parafovea than in the fovea1 avascular zone. In a previous study, we provided evidence that spatial variations in melanin and oxyhemoglobin absorption have influenced earlier reflectometric measurements of macular pigment (Alexander, Kilbride, Fishman & Fishman, 1987). In the present report, we describe a method for assessing the spatial distribution of macular pigment in normal subjects, using a television-based, imaging fundus reflectometer, in which the presumed effects of melanin and oxyhemoglobin are minimized. In this procedure, a digitized fundus image obtained at an illuminating wavelength of 559 nm (governed primarily by melanin and oxyhemoglobin absorption) is subtracted from a digitized image obtained at 462 nm (determined by macular pigment absorption as well as by melanin and oxyhemoglobin) to obtain the spatial variation of the double density of macular pigment. The 559 nm image is corrected for the change in melanin and oxyhemoglobin density between 559 and 462 nm. We have applied this analysis to seven normal individuals to deter-
mine the degree of intersubject variability in the double density of macular pigment among this group. METHOD
Subjects
Seven individuals with normal vision participated in the study. All were Caucasian with a best-corrected Snellen visual acuity of 20120 or better and normal color vision as assessed with a Nagei anomaloscope. The mean age of the subjects was 33 yr; the range was 22-39 yr (Table I). Older subjects were excluded to minimize the possibility of lens scatter (Sigelman, Trokel & Spector, 1974). Informed consent was obtained from all subjects after the nature of the procedure had been explained fully. Apparatus
The television-based fundus reflectometer used to measure fundus reflectance has been described previously (Kilbride. Read, Fishman & Fishman, 1983). Briefly, a Sierra Scientific video camera containing an RCA Type 4804 silicon intensifier target (SIT) camera tube viewed the fundus through the optics of a modified Zeiss fundus camera. A fiberoptic light guide illuminated by a computer-controlled optical system replaced the normal Zeiss light source. In this optical system, light from a 150-watt xenon arc lamp passed successively through an infrared blocking filter; a neutral density wedge and an interference fiber wheel, both of which were driven by stepping motors; and an electromechanical shutter. The spectral characteristics of the measurement light were defined by 13 interference filters with peak wavelengths that ranged from 462 to 697 nm in approx. 20 nm steps and half-height bandwidths of approx. 10 nm. The output of the video camera was digitized by a high-speed analog-todigital converter, with an image resolution of 112 by 100 pixels (each pixel subtended 0.1” of visual angle) and 256 levels of gray scale resolution. The digitized images were stored on a magnetic disk and were later transferred to magnetic tape for archival storage. Procedure
The procedure used to acquire the fundus images was identical to that described previously for studies of cone pigment density differences (Kilbride et al., 1983). TO provide calibrations for the fundus reflectance of the
665
Image analysis of macular pigment Table 1. Subject ages, parameters of individual macular pigment distributions
(1) Subject No.
(2)
Mean SD
is)
(7) e(o) Vertical
(8) r.m.s. Error Horizontal
(9) r.m.s. Error Vertical
Sex
Age
M
22 30 31 36 36 37 39
0.37 0.35 0.33 0.22 0.26 0.30 0.44
0.40 0.36 0.33 0.27 0.29 0.35 0.45
0.85 1.10 I .03 1.06 0.78 0.87 1.37
0.87 0.89 0.92 0.98 0.81 0.93 1.17
0.03 0.03 0.02 0.03 0.02 0.03 0.02
0.04 0.03 0.03 0.03 0.03 0.04 0.03
33 5.8
0.32 0.07
0.35 0.06
1.01 0.20
0.94 0.12
0.03 0.01
0.03 0.01
a$, Vertical
eye, an artificial eye with a focal length of 24 mm and with an imaging surface of Halon powder was scanned prior to subject testing. The spatial distribution of light in the digitized image of the artificial eye was used to correct the digitized fundus images for spatial nonuniformities in the radiometric response of the imaging system, while the reflectance values of the artificial eye allowed comparisons of fundus reflectances among subjects. The pupil of the test eye was dilated and accommodation paralyzed with 2.5% phenylephrine hydrochloride and 1% cyclopentolate hydrochloride drops. Head position during testing was maintained by a dental impression bite block and forehead rests; the nontested eye fixated upon a dimly illuminated target. The eye was exposed for 30 set to a 605 nm bleaching light of 6.12 log td, which is sufficient to bleach more than 97% of the middle- and long-wavelength cone pigments (Alpern, Maaseidvaag & Ohba, 1971). Immediately after the offset of the bleaching light, a series of digitized images of the bleached fundus was obtained with illumination provided by monochromatic light from each of the 13 interference filters. The exposure duration of each wavelength was 83 msec (5 video fields), during which time a fundus image was electronically integrated on the camera tube. The interval between exposures was approx. 220 msec, and each complete wavelength series was obtained in about 4 sec. In the present study, either two (for 2 subjects) or three (for 5 subjects) complete wavelength series were obtained, with each series preceded by a bleaching exposure. The computer adjusted the light radiance at each wavelength so that the camera operated near the center of its linear range. This procedure was necessary since the output of the video camera is linear only within an input range of 10 : 1, while fundus spectral reflectance varies by more than 100: 1. The measurement human
(6)
(4) 6’ Hori.%rtal
F M F M F M
(3)
a(o) Horizontal
lights produced minimal bleaching of the middle- and long-wavelength cone photopigments and were considerably below the maximum permissible exposure (MPE) for retinal irradiance. The highest retinal illuminance was 4.57 log td-set (at 559 nm), which would bleach approx. 0.7% of cone photopigments in the dark-adapted eye (Alpern et al., 1971). The highest retinal irradiance was 2.47 x 10m4W cme2 (at 462nm), which is more than 4 log units below the MPE at our exposure duration (Delori, Parker & Mainster, 1980). For each subject, each of the digitized images of the fundus was aligned with a selected anchor image to compensate for any small eye movements that may have occurred between the periods of fundus illumination. To accomplish the image registration, the fundus image at a given wavelength was alternated rapidly with the anchor image on a video monitor. Using a trackball system, the operator moved the video image of the fundus until apparent movement of retinal detail was minimized. As a result of this image alignment, the reflectance factors of corresponding retinal regions could be compared across wavelengths. For each subject, the multiple fundus images obtained at each wavelength were averaged to provide a mean digitized fundus image for that subject at that wavelength. In the following image analysis, as in previous studies (Brindley & Willmer, 1952; Van Norren & Tiemeijer, 1986), reflectance factors were obtained. The reflectance factor [R,, (i.)] of each pixel (coordinates x and y) represents the ratio of the radiant intensity of that pixel in the digitized image of a human fundus [Z,l,Y,,.j,] to the radiant intensity of the corresponding pixel in the digitized image of the artificial eye [ZC,,Y,,,j,]:
(2)
PAUL E. KILBRIDE et al
666 RESULTS
An individual series of digitized images of the bleached fundus of a typical normal subject, obtained with monochromatic illumination, is presented in Fig. 1. At short wavelengths of illumination (top row) there is a region of low reflectance within the fovea that is not apparent at longer wavelengths (bottom row). The spatial and spectral characteristics of this region are suggestive of macular pigment. However. we have reported previously that macular pigment is not the sole determinant of this shortwavelength reflectance pattern (Alexander et al., 1987). The following analysis indicates the validity of this proposal for the present group of normal subjects. Our analysis is similar to that of Van Norren and Tiemeijer (1986) in which macular pigment, lens pigment, melanin, and oxygenated hemoglobin are considered to be the primary determinants of the reflectance of the bleached fundus. All of these pigments have some degree of absorption at short wavelengths. In accordance with Van Norren and Tiemeijer (1986) we assume that these pigments obey Beer’s law and are located in front of a spectrally neutral reflector (sclera), the properties of which are the same between the fovea and parafovea. Figure 2 presents the density spectra of these pigments, which, except for oxyhemoglobin, are plotted directly from published values. The density spectra for macular pigment and the lens were obtained from Wyszecki and Stiles (1982) while that for melanin is replotted from Hunold and Malessa (1974). The density spectrum for oxyhemoglobin is 0.02 times the values of Mook et al. (1969) which approximates the optical
Wavelength
3 Wavelength
(nm)
Fig. 4. Mean log spectral reflectances of the fovea and parafovea of the seven normal subjects. Error bars indicate ) 1SEM.
density of oxyhemoglobin in the parafovea of the human fundus (cf. Van Norren & Tiemeijer, 1986). To demonstrate the contribution of these ocular pigments to reflectance differences between the fovea and parafovea, we first determined the average spectral reflectance of two windows within the fundus image, as depicted in Fig. 3. The fundus image in this figure was obtained at 462 nm, and is identical to that illustrated in Fig. 1. The central window, indicated by the circle, is 0.7” in diameter. The outer window, indicated by the ring, has an inner diameter of 6” and an outer diameter of 7 I. This latter value was the limit of the common area for the 244 fundus images obtained from the seven subjects. The mean log spectral reflectances of these two windows are shown in Fig. 4. In agreement with previous studies (Brindley & Willmer, 1952; Van Norren & Tiemeijer, 1986; Kilbride, Fishman, Hutman & Fishman, 1986), the log reflectances of both the fovea and parafovea are lowest at short wavelengths and increase with increasing wavelength. Moreover, there is a systematic difference between the mean log spectral reflectances of the fovea and parafovea, as reported previously (Brindley 8t Willmer. 1952; Van Norren & Tiemeijer, 1986) with the greatest difference at short wavelengths. We have plotted this difference [p(k)], which is equivalent to the log ratio of parafoveal to fovea1 reflectance factors, in Fig. 5:
(run)
Fig. 2. Density spectra of macular pigment, lens pigment, melanin, and oxyhemoglobin.
(3
Fig. 1. Digitized fundus images of a typical normal subject obtained with the following wavelengths of illumination (left to right): 462, 480, 502, 521 nm (top); 541, 559, 580, 598 nm (middle); 617, 634, 657, 675 nm (bottom). An additional image, obtained at 697 nm and essentially identical to that at 675 nm, was omitted for lack of space. (Due to limitations inherent in the reproduction process, these photographs do not accurately represent the actual digitized fundus images.)
Fig. 3. Digitized fundus image obtained at 463 nm of illumination (from Fig. I). The circle and ring illustrate the retinal regions whose pixel values were averaged to obtain estimates of fovea1 and parafoveai spectral reflectance, respectively. 667
669
Image analysis of macular pigment
---
0.5
0.4
_____.
~
r.
500
-
-._
-
550 Wavelength
MaMCulllrPioment Melanin “WlO~bbt”
camblned
600
650
700
(nm)
Fig. 5. Mean log reflectance ratios @(A)] between the parafovea and fovea. The solid line is a least-squares fit of the combined density spectra of macular pigment, melanin, and oxyhemoglobin. The interrupted lines show the constituent density spectra of the individual ocular pigments. Error bars indicate If: 1SEM.
Since the greatest values of p(I) occur at short wavelengths, previous studies (Brindley & Willmer, 1952; Van Norren & Tiemeijer, 1986) have presumed that p (A) is due only to macular pigment absorption. However, the presence of nonzero values of p (A) at long wavelengths, also seen in previous studies (Brindley & Willmer, 1952; Van Norren & Tiemeijer, 1986), suggests that macular pigment is not the only contributor. To illustrate the relative contribution of ocular pigments besides macular pigment to the p(A) values, we used a least-squares procedure to fit the data in Fig. 5 by the combined density spectra of macular pigment, melanin, and oxyhemoglobin:
where Di(n) represents the density spectrum of melanin, macular pigment, or hemoglobin, respectively, and a, are derived weighting coefficients. In this analysis, we assume that lens absorption is the same for both retinal regions, since the measurement light passes through the crystalline lens in a focused beam. The solid line in Fig. 5 represents the best-fit combined density spectrum. The squared multiple regression coefficient (mr2) was 0.98; the standard error of the estimate was 0.04. The interrupted lines represent the values of aiDi for the individual pigments. At wavelengths longer than approx. 530 nm, melanin and oxyhemoglobin appear to be the major determinants of p(l). At short wavelengths, p (J.) is apparently determined
jointly by macular pigment and melanin, with a minor contribution from oxyhemoglobin. Therefore, spatial variations in macular reflectance at short wavelengths do not represent the distribution of macular pigment alone. The following analysis describes a method for reducing the effects of melanin and oxyhemoglobin absorption on reflectometric estimates of macular pigment double density. We first apply this analysis to the mean data of the seven subjects, and then present results from individual subjects. Figure 6a plots the log reciprocal reflectance factor (log [ 1/R.,,:,,,]) of each pixel in the mean image of the seven subjects, obtained at 462 nm. To reduce pixel noise, the mean values in this figure and in Fig. 6b were linearly filtered by a moving average filter, in which each pixel is replaced by an average of its neighbors (a 3-by-3 array with equal weight for each pixel). As noted above, the spatial pattern of reflectance at 462 nm is not due entirely to macular pigment; melanin and oxyhemoglobin also contribute. Therefore, to determine the spatial variation in macular pigment alone, it is necessary to remove the influence of melanin and oxyhemoglobin from the data in Fig. 6a. This can be accomplished by subtracting a fundus reflectance distribution obtained at a wavelength that represents predominantly melanin and oxyhemoglobin absorption. It is evident from the data in Fig. 5 that wavelengths longer than 530 nm can provide such a distribution. It is also necessary that the change in the extinction coefficient between 462 nm and the chosen wavelength be equivalent for both melanin and oxyhemoglobin. The reflectance data at a wavelength of 559 nm fulfill these two criteria. The mean log reciprocal reflectance factor of each pixel at 559 nm (log ( I/Rr.r.559])is presented in Fig. 6b. This surface was multiplied by the average change in the extinction coefficients of melanin and oxyhemoglobin (1.14) that occurs between 559 and 462 nm. Finally, the product was subtracted from the data of Fig. 6a to derive the corrected double density [6&J of macular pigment:
- 1.14*loisWRx,y,d
(5)
It should be noted that lens density was not included in this analysis. Absorption by the lens adds a uniform constant to each reflectance
670
PAUL E. KILBRIDEet al.
surface but does not affect the shape of the surface. Moreover, the change Ott lens density between 462 and 559 nm, for the age range of our subjects, is not of sufficient magnitude to alter the results significantly- iwyszecki and Stiles. 1982). The result of thus image subtraction is shown in Fig. 6~. According to our analysis, this surface represents the spatial variation in the double density of macular pigment across the central fovea. To examine the symmetry toi the surface shown in Fig. 6c, we have plotted isodensity contours in Fig. 7. The spatial variation of a;,; is approximately symmetrical. To illustrate further the spatial distribution of ilk) and to indicate the degree of intersubject variability, we have plotted cross-sections of Fig. 6c, measured along the horizontal and vertical meridians, in Fig. 8a and b, respectively. To obtain these cross-sections, the image analysis described above (equation 5) was applied separately to each individual subject, and values of S& for each subject were obtained along the horizontal and vertical meridians. Each point in a crosssection of an individual subject represents an average of a region 3 pixels wide centered on that point and orthogonal to the measured meridian. The hatched regions in Fig. 8a and b represent the mean cross-sections of the seven subjects + I SEM, measured along the horizontal and vertical meridians, respectively. The solid lines in Fig. 8 represent Gaussian functions of the form:
Fig. 6. Mean log reciprocal of fundus reflectance at 462 nm (a); 559 nm (b): and 462 nm after correction for absorption by melanin and oxyhemoglobin (c). See text for details.
where u and h are scaling parameters, x indicates eccentricity, and 1~and D are the mean and standard deviation of the dtstribution, respectively. This equation was tit to the mean data for each of the two meridians by an iterative curve-fitting program with a leastsquares criterion. Values of p and (T for the mean data were 0.13 and 0.99’ for the horizontal meridian, and 0.09 and 0.94 for the vertical meridian. For both meridians, the r.m.s. error was 0.01. As a second measure of individual variation, Gaussian functions were also ht separately to the horizontal and vertical meridians for each of the individual subjects. An estimate of a&,, at the fovea1 center for each subject for each meridian was obtained from the difference between the value of 0 & at the peak of the best-fit Gaussian function and the value of ~3;~~ at the asymptotic
Image analysis of macular pigment
4-3~ .,,,,, ~
),,,(,
-3
-2
-1
0
1
2
3
Degrees
Tem#mnl
1
HamI
Fig. 7. Isodensity contours of the surface shown in Fig, 6c.
level. These values of 6;,, for the horizontal and vertical meridians are given in Table 1 (columns 4 and 5, respectively). Values of c for the individual subjects, representing the spatial variation in S&, along the horizontal and vertical meridians, are also presented in Table I (columns 6 and 7, respectively). The r.m.s. errors of the best-fit Gaussians are given in Table I (columns 8 and 9, respectively).
Discussion
The spatial variation in the double density d&s of macular pigment that we have derived from imaging fundus reftectometry (Figs 6-8) is similar to previous measures obtained within the human retina by psychophysical methods (Stabell & Stabell, 1980; Pease & Adams, 1983; Vienot, 1983; Moreland & Bhatt, 1984;
671
Werner, Donnelly & Kliegel, 1987) and within the monkey retina by microdensitometry (Snodderly et al., 1984b). In those studies, the macular pigment absorbance was highest in the fovea1 center and declined to a low constant level by approximately 4” of eccentricity. In addition to estimates of macular pigment absorption, our data also provide an indication of spatial variations in melanin absorption within the macula. Melanin is a screening pigment within the retinal pigment epithelium and choroid that may have a protective antioxidant function (Ostrovsky, Sakina & Dontsov, 1987). A reduction in melanin, for example, has been implicated in the development of retinal disorders such as age-related macular degeneration (Weiter et al., 1985). Within the choroid, the density of melanin is highest in the fovea1 center (Weiter et al., 1986). Within the pigment epithelium, melanin density tends to be higher in the far periphery than in the macular region (Schmidt & Peisch, 1986), but there is an absorption peak within the central fovea (Weiter et al., 1986). As a consequence, the combined melanin density of the pigment epithelium and choroid is greatest in the fovea1 center. Our measurements are consistent with these previous reports. The reflectance data at 559 nm, shown in Fig. 6b, represent prima~ly the spatial variation in melanin absorption. There is a peak absorption in the fovea1 center, in agreement with opticai measurements in autopsy eyes (Weiter et al., 1986). Our estimates of the double density of macular pigment within the central fovea are lower than values previously obtained by fundus reflectometry (Brindley & Willmer, 19.52; 0.4
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iemporai
‘
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11
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Eccentricity(Degf
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Fig. 8. The spatial pattern of macular pigment absorption at 462 nm across the horizontal (a) and vertical (b) meridians of the visual field. The hatched regions show means i: i SEM. The solid lines represent Gaussian functions fit by a least-squares procedure to each of the two meridians.
672
PAUL E. KILBRIDEet al
Van Norren & Tiemeijer, 1986). This is due primarily to the fact that we have attempted to eliminate the contribution of melanin and oxyhemoglobin in our analysis. In previous studies (Brindley & Willmer, 1952; Ishak et al., 1967; Van Norren & Tiemeijer, 1986), values of p(%), representing the difference between the Iog reflectances of the parafovea and fovea, were used as a measure of macular pigment density. However, our results indicate that shortwavelength reflectances should be corrected for melanin and oxyhemoglobin absorption before accurate estimates of macular pigment double density can be derived. Since our values of p (i.) are comparable to those of earlier reports (Brindley & Willmer, 1952; Van Norren & Tiemeijer, 1986), our estimates of S;,, are necessarily lower than in those studies. Our macular pigment double densities are also lower than might be expected from psychophysical studies, in which optical densities average approx. 0.5 (e.g. Werner et al., 1987; Wyszecki & Stiles, 1982). Since there appears to be considerable intersubject variability in psychophysi~lly measured macular pigment density (Werner et al., 1987; Pease, Adams & Nuccio, 1987). it is possible that our subjects represent the lower range of the distribution. However, it is more likely that the apparent discrepancy results from fundamental differences between psychophysical and reflectometric methods. One of the primary factors is light scatter, which is an unavoidable influence on reflectometric estimates of the double density of macular pigment, as well as of photoreceptor photopigment double density (Rushton, 1965; King-Smith, 1973; Margolis, Siegel & Ripps, 1987). While a fraction of the light reaching the measuring instrument has passed twice through the layer of macular pigment, an unknown proportion has passed through only once, or not at all. Some of the light may have been scattered within the retina or reflected specularly from the internal limiting membrane, particularly in the eyes of younger subjects (Delori, Gragoudas, Francisco & Pruett, 1977). In addition, light from the measuring beam may have been back-scattered from the cornea and/or lens or forward-scattered in its return through the lens center (Sigelman et al., 1974). All of these factors would tend to reduce the contrast of the fundus images and could therefore result in a lower estimate of macular pigment absorption than would be expected from psychophysical studies. Moreover, refiectometric estimates of
macular pigment spatial distribution may be inff uenced by the optical properties of the fovea1 pit (Williams, 1980). While light scatter poses a problem for reflectometric estimates of the spatial distribution of macular pigment, the use of psychophysical procedures to assess spatial variations in macular pigment atso has potential limitations. Psychophysical studies measure the screening effect of macular pigment on light that has been absorbed by cone photoreceptors. As a consequence of the Stiles&?rawford effect. light that is of normal incidence tfn the photoreceptors is more likely to be absorbed than light that is scattered within the retina (Crawford, 1972). Since macular pigment is primarily contained within the inner retimi (Snodderly et al., 1984a), the psychophysically measured optical density of macular pigment represents primarily the density of pigment interposed between the individual photoreceptors and the exit pupil of the eye. However, evidence indicates that the psychophysically measured density of macular pigment represents the density at the edge of a test flash. rather than an average of the stimulated region (Werner et al., 1987). Consequently, there could be a considerable reduction in the density of macular pigment within specific regions of the fovea without necessarily a decrease in the psychophysically measured optical density of macular pigment. The optimum procedure for the psychophysical measurement of the spatial distribution of macular pigment would be the presentation of a tiny test flash at a series of retinal locations (e.g. Williams, MacLeod & Hayhoe, 1981), a timeconsuming procedure requiring precise fixation. Consequently. despite the limitations of light scatter. our method for assessing macular pigment by imaging fundus reflectometry can potentially provide information about spatial patterns of macular pigment within the intact human retina, both normal and abnormal, that is not obtained easily by alternative methods. Moreover, the method has potential application to the study of macular pigment distribution in animal retinas as well. Rcknowfedgements--Theauthors thank Morton F. Goldberg and Neal S. Peachey for helpful comments on the manuscript, Robert J. Anderson for statistical advice, and Kathleen Louden for editorial assistance. The research was supported by NE1 Grants EY04848, EYO6589, Core Grant EY01792, and a center grant from the National Retinitis Pigmentosa Foundation Fighting Blindness, Baltimore. MD.
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