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.
Scanning laser ophthalmoscopy is a confocal imaging technique that allows high-contrast imaging of retinal structures. Rapid, involuntary eye movements during image acquisition are known to cause artefacts and high-speed imaging of the retina is crucial to avoid them. To reach higher imaging speeds we propose to illuminate the retina with multiple parallel lines simultaneously within the whole field of view (FOV) instead of a single focused line that is raster-scanned. These multiple line patterns were generated with a digital micro-mirror device (DMD) and by shifting the line pattern, the whole FOV is scanned. The back-scattered light from the retinal layers is collected via a beam-splitter and imaged onto an area camera. After every pattern from the sequence is projected, the final image is generated by combining these back-reflected illumination patterns. Image processing is used to remove the background and out-of-focus light. Acquired pattern images are stacked, pixels sorted according to intensity and finally bottom layer of the stack is subtracted from the top layer to produce confocal image. The obtained confocal images are rich in structure, showing the small blood vessels around the macular avascular zone and the bow tie of Henle's fiber layer in the fovea. In the optic nerve head images the large arteries/veins, optic cup rim and cup itself are visualized. Images have good contrast and lateral resolution with a 10°×10° FOV. The initial results are promising for the development of high-speed retinal imaging using spatial light modulators such as the DMD.