2.1.

Macular Pigment Reflectometer

The optical arrangement is shown in Fig. 1 . Light from a 30-W halogen lamp is delivered at the eye through three optical relay systems. The first relay (L1, L2) images the filament of the lamp on a mask. Between L1 and L2, filtering (GG395, Schott, Mainz, Germany) occurs to cut off uv. This to reduce the level of dangerous light in subjects with their eye lenses removed. The second relay (L3, L4) images the filament within the mask on a small mirror $M$ at $45\mathrm{deg}$ . The last relay (L5, L6) forms an image of the eye pupil at the plane of $M$ , where the illumination beam coming from $M$ , and detection beam, passing over $M$ are separated (“pupil separator”). The relay (L5, L6) forms a Badal system, as L6 is movable to focus a stop (between L3 and L4) at the retina. Focusing range is from $-15\mathrm{D}$ to $+15\mathrm{D}$ . The resulting retinal spot has a diameter of 1-deg visual field, a retinal illuminance of $1.04\times {10}^7\mathrm{Td}$ , and a retinal irradiance of $4.6\mathrm{mW}∕{\mathrm{cm}}^2$ . The calculated maximum exposure time, based on the recommendations of the Health Council of the Netherlands (1993)¹⁵ is $26\min$ ; for people with aphakia, it is $20\min$ . The detection channel has the Badal system (L5, L6) in common with the illumination system, with no overlap in the lenses itself to keep instrumental stray light to a minimum. The pupil separator contains masks to limit the illumination and detection configuration (separation $0.8\mathrm{mm}$ ) in the pupil to a 3-mm circle (Fig. 1). For subjects with a pupil diameter smaller than $3\mathrm{mm}$ , absolute calibration of percentage reflection no longer holds. However, this does not immediately make accurate measurements of macular pigment impossible, as these are determined from the shape of the spectral reflection. A diameter of $2.3\mathrm{mm}$ is about the practical lower limit. Lenses L7 and L8 form an image of the retinal spot on the tip of an optical fiber. A mask at the tip of the fiber defines a diameter of $1\mathrm{deg}$ at the retinal plane. The fiber is the receiving part of a commercial fiber spectrometer (Ocean Optics SD2000, Ocean Optics Inc. Dunedin, Florida). Optical resolution is $5.8\mathrm{nm}$ (FWHM); the range 400 to $800\mathrm{nm}$ was used for analysis. The output of the spectrometer is sent to a personal computer. Integration time was set at $1\mathrm{s}$ . The displayed reflection spectrum is the mean and standard deviation of two succeeding 1-s periods.

Fig. 1

Optical scheme of the MPR. An image of the filament of the halogen lamp is relayed by lenses L1 to L4 to a mask and then to a mirror $M$ . Filters (GG395, Schott) are inserted to reduce uv light. The Badal system L5 to L6 has L6 as a movable lens to focus a retinal stop (between L3 and L5) on the retina. The detection channel has the Badal system in common with the illumination system, without overlap of the light paths in lenses. Lenses L7 to L8 form an image of the retinal field on the tip of a fiber that connects to a commercial spectrometer. The insert shows the dimensions of the illumination and detection beams at the level of the pupil.

Reflectance was calibrated with a surface painted with Eastman 6080 white at a distance of $230\mathrm{mm}$ from the pupil plane position (”white calibration”). The surface was mounted at the end of a black anodized tube to eliminate room light. The Eastman 6080 white surface was compared with a ${\mathrm{BaSO}}_4$ surface. Differences were accounted for in the software. During the calibration, the spectrometer averaged 30 measurements of $1\mathrm{s}$ each. The first lens was set to a position such that the white surface was conjugate with the retinal planes of the instrument. Because the distance of $230\mathrm{mm}$ from the pupil plane is 10 times the focal distance of the eye, the measured spectrum equaled a 1% reflection from the eye. This reflection calibration accounts both for the spectral sensitivity of the detection system, and for the spectral content of the illumination. The white calibration was corrected for spurious reflections in the front lenses by averaging 30 measurements with a light trap at the position of the eye (“dark calibration”). The light trap consisted of two polished black plates at a tapering angle, positioned at the pupil plane of the instrument. The dark calibration was taken with the lens position optimized for each subject (see Sec. 2.3). Wavelength was calibrated with a lamp containing mercury and argon lines (Avalight-CAL, Avantes Eerbeek, The Netherlands).

2.2.

HFP

We used a portable instrument developed by Mellerio ¹⁶ In summary, a test field flickers between a blue light that is highly absorbed by the macular pigment (MP) and a green light that is not absorbed by the MP. A minimum flicker match is made by adjusting the intensity of the blue light when the retinal image of the 1-deg test field lies on the fovea. Another match is made away from the fovea, where the MPOD is assumed to be negligible. The test field was an annulus of 5-deg inner radius and of 1-deg width, centered on the foveal fixation spot. The logarithm of the ratio of the blue luminosity for the foveal match to that for the extramacular match gives the MPOD.

2.3.

Protocol

The research followed the tenets of the Declaration of Helsinki and was approved by the local medical ethics committee. All subjects ( $n=20$ , ages 18 to 79 years) were Caucasian, not known to have an eye disease, and with a visual acuity of at least 0.8. The purpose was explained at the beginning of the experiment, and written informed consent was obtained. Chin rest and temple pads were used to help maintain head position. The instrument was aligned using a joystick with the subject’s eye. The (clearly visible) illumination beam was set in the subject’s pupil a little above its center, allowing room for the invisible detection beam. The subject was asked to fixate the center of the 1-deg spot. Initial focusing was obtained with a diopter scale on the instrument using the spectacle prescription. Next the subject was asked whether the spot edges were sharply focused. If necessary, an adjustment was made. Correcting refraction resulted in concentric illumination and detection fields at the retina. Without focusing, they are concentric posterior or anterior to the retina, and reflected light is lower in amplitude. Therefore, a final adjustment was made by optimizing the amplitude of the measured reflection spectrum. The experimenter viewed a reflectance curve, updated once per second, together with the highest curve (with maximum reflectance between 500 to $600\mathrm{nm}$ ) obtained since the start of the measurement. This feature also facilitated optimizing the position of illumination and detection beam with regard to the subject’s pupil. When the front lens was optimized, the subject leaned back, allowing the experimenter to perform the dark calibration. Thereafter, final alignment took place. Dark calibration changed very little over the course of hours, thus in fact it could have been taken less frequently.

Reflections from the corneal surface and/or lens were minimized by adjusting the distance between eye and the instrument, and minor adjustments in the position of the illumination beam. Corneal reflections show up as a relatively high signal at the short wavelengths compared to the middle wavelengths. We estimated that alignment time was sufficient to bleach more than 95% of the visual pigments. Subjects were asked to occasionally blink but keep their eyes wide open during measurements. Five spectra were measured and stored on a disk for further analysis in three conditions: (1) natural pupil, dark room, for getting statistics on measurements with natural pupil in an optimal environment; (2) natural pupil dim room light, for getting statistics on measurements with natural pupil in a less optimal environment with a disturbing light, in this case from a dimmed incandescent lamp set at 3 lux at the eye; (3) dilated pupil, dark room, for having an optimal reference with no disturbing room light and no possible influence of interception of measurement light by the pupil edges. Ten subjects were remeasured in condition 1 after an interval of at least 3 days to check reproducibility.

With the HFP instrument, according to the prescribed protocol, also five measurements were taken.

2.4.

Data Analysis

A slightly modified model of pathways of light in the eye was used to analyze the spectral data.¹⁷ Briefly, the model contains three reflectors, the inner limiting membrane (ILM), the cone receptor disks, and the choroidal space. Absorption anterior to the receptor layer takes place in the lens and in the macular pigment. Absorption posterior to the receptor layer is in melanin and blood. Absorption in visual pigment is neglected because of bleaching by the high light levels. The retinal area of $1\mathrm{deg}$ to be analyzed was considered homogeneous in macular pigment and melanin content, although we recognize that this is a simplification. A more detailed description of the model is in the appendix.

The Levenberg-Marquardt routine¹⁸ was used to fit the data with the model by minimizing chi squares. The wavelength range used was from 400 to $800\mathrm{nm}$ . All parameters were allowed to vary simultaneously. For a single measurement, the noise in the data points was assumed to originate mainly from photon noise, with a small addition from head or eye movements of the subject. The standard deviation in the spectral data points were therefore calculated from two succeeding 1-s periods, and were used to weight the data points in the fitting process. For a typical example of this noise, see the error bars in Fig. 2 in Sec. 3. A single measurement containing relatively large eye movements was easily recognized by a large standard deviation at the long wavelength points and was generally rejected and replaced immediately during the measurements.

Fig. 2

Typical reflectance spectrum exhibiting high reflectance at long wavelengths, with a gradual decrease caused by absorption in melanin and blood. At about $550\mathrm{nm}$ , the MP causes a sharp decline, followed by a plateau between 500 and $450\mathrm{nm}$ . Thereafter, another sharp decrease is caused by absorption in the crystalline lens. A best fitting model curve is drawn through the data points. The error bars show the standard deviation in the data points. The same model curve, but with the absorption of MP set to zero, is shown as a dashed line. The absorption curve of MP from literature (Ref. 31), as used in the model, is shown at the bottom (see the scale on the right).