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Retinal blood vessel oxygenation is considered to be an important marker for numerous eye diseases. Oxygenation is typically assessed by imaging the retinal vessels at different wavelengths using multispectral imaging techniques, where the choice of wavelengths will affect the achievable measurement accuracy. Here, we present a detailed analysis of the error propagation of measurement noise in retinal oximetry, to identify optimal wavelengths that will yield the lowest uncertainty in saturation estimation for a given measurement noise level. In our analysis, we also investigate the effect of hemoglobin packing in discrete blood vessels (pigment packaging), which may result in a nonnegligible bias in saturation estimation if unaccounted for under specific geometrical conditions, such as subdiffuse sampling of smaller blood vessels located deeper within the retina. Our analyses show that using 470, 506, and 592 nm, a fairly accurate estimation of the whole oxygen saturation regime [0 1] can be realized, even in the presence of the pigment packing effect. To validate the analysis, we developed a scanning laser ophthalmoscope to produce high contrast images with a maximum pixel rate of 60 kHz and a maximum 30-deg imaging field of view. Confocal reflectance measurements were then conducted on a tissue-mimicking scattering phantom with optical properties similar to retinal tissue including narrow channels filled with absorbing dyes to mimic blood vessels. By imaging at three optimal wavelengths, the saturation of the dye combination was calculated. The experimental values show good agreement with our theoretical derivations.
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