Dual-Wavelength Autofluorescence Imaging for Macular Pigment Quantification

Macular pigment imaging by dual wavelength AFI was performed on a Heidelberg MultiColor Spectralis as previously described.^{11 (link)} After pupil dilation, the subject's macula was raster scanned over 30° centered on the fovea by alternating blue and green laser light (485.6 and 516.7 nm, respectively) for approximately 30 seconds while AFIs of RPE lipofuscin for each excitation wavelength were collected and averaged. Autofluorescence detection was restricted to wavelengths above 530 nm with the help of a barrier filter. Specialized software then performed digital subtraction of the green excitation AFI from the blue excitation AFI using a correction factor to account for the fact that the blue excitation is not quite at the peak of macular pigment absorption (460 nm) and that there is still a substantial amount of macular pigment absorbance with the green excitation. The instrument's effective extinction coefficients, K_mp(Λ), are 0.789 for 485.6 nm and 0.205 for 516.7 nm, and the correction factor is: 1/[K_mp(485.6) − K_mp(516.7)] = 1.71, based on the image processing method described by Delori and colleagues^{32 (link)} using the macular pigment extinction coefficients calculated by Stockman and Sharp.^{33 (link)} In order to compensate for background signal, an offset parameter (“OFF”) is subtracted. This value is recorded by the system internally during the acquisition of the blue/green AFI with the lasers turned off.
A subtracted macular pigment autofluorescence attenuation image is produced showing a white region centered on the fovea corresponding to the macular carotenoid pigment (Fig. 1). The instrument calculates the average MPOD, SD, and range of MPOD levels along a series of concentric one-pixel width circles. The results are then plotted on a graph from 0° to 15° with a red curve corresponding to the average MPOD at each eccentricity, a green region corresponding to the SD of the average MPOD, and a blue region corresponding to the high and low range of MPOD. The user must choose a reference eccentricity where the MPOD is defined as zero. We chose 9.0° because the vast majority of subjects had near baseline measurements at this distance from the fovea, and readings beyond this eccentricity would likely be affected by retinal vasculature or the optic nerve, typically manifested as an increase of SD and range at eccentricities beyond 9°. For the instrument's automated results table, the user not only selects the zero point radius (green vertical line; 233 pixels at 9°; “plateau” column on the report) but can also choose two other analysis eccentricities. We routinely used 0.5° (red vertical line; 12 pixel radii at 0.47°) and 2° (blue vertical line; 51 pixel radii at 1.99°). The most important parameters from the report that we used for our analyses were the “average OD on radius,” which we report as “MPOD X°” (macular pigment optical density at X°) corresponding to the 360° averaged MPOD at that particular radius/eccentricity and “OD sum of volumes,” which we report as “macular pigment volume under the curve at X°” (MPVUCX°), which is the integral of the total MPOD within X° of the fovea and should correspond to the total macular pigment within that particular region always using 9° as the reference eccentricity.