Captured images were corrected for systematic variations in the spectral sensitivity of the system with a correction factor derived from images of a white surface with a flat reflection spectrum. The corrected monochromatic images were then aligned assuming that degradation due to eye movement can be approximated by a combination of translation, rotation, and scaling.
22 Alignment was subjectively reviewed and repeated if movement artifacts remained.
Using a Beer-Lambert law model
19 –21 (equation 1) we then calculated our metric differential light absorption (DLA) on a pixel-by-pixel basis from the aligned images. DLA is a measure of the relative wavelength-specific, light-absorption properties of the imaged tissue, which is dependent on the concentration of wavelength-specific absorbers in the tissue. Specifically, we find the least-squares solution to equation 1 in which the constants
a and
b are the molar extinction coefficients of oxygenated and deoxygenated hemoglobin in water,
18 covariate ω is a second absorber inversely related to DLA.
I λ and
I 570nm represent the pixel intensities of the images obtained at each wavelength and at 570 nm, respectively. By calculating DLA on a pixel-by-pixel basis we can produce spatial maps of DLA by the imaged tissue.
Similar approaches have been used to estimate oxygen saturation in animal brain tissue
19 –21 and human retinal vessels
8 –10,13 –15 using instruments calibrated manometrically or by oxygen-breathing experiments. Because of the similarities between our methods and those of previous studies, we expect that our DLA metric is related to oxygen saturation. However, variations in the reflective characteristics of individual eyes make accurate in vivo calibration to oxygen saturation extremely difficult. Since in the present study we are solely concerned with determining the presence or absence of correlations with VF sensitivity, we conservatively report DLA in arbitrary relative units.