Quantification of optical lensing by cellular structures in the living human eye.

Cells and other microscopic phase objects can be visualized in the living retina, non-invasively, using non-confocal light detection schemes in adaptive optics scanning light ophthalmoscopes (AOSLOs). There is not yet widespread agreement regarding the origin of image contrast, nor the best way to render multichannel images. Here, we present data to support the interpretation that variations in the intensity of non-confocal images approximate a direct linear mapping of the prismatic deflection of the scanned beam. We advance a simple geometric framework in which local 2D image gradients are used to estimate the spherocylindrical refractive power for each element of the tissue. This framework combines all available information from the non-confocal image channels simultaneously, reducing noise and directional bias. We show that image derivatives can be computed with a scalable, separable gradient operator that minimizes directional errors; this further mitigates noise and directional bias as compared with previous filtering approaches. Strategies to render the output of split-detector gradient operations have been recently described for the visualization of immune cells, blood flow, and photoreceptors; our framework encompasses these methods as rendering astigmatic refractive power. In addition to astigmatic power, we advocate the use of the mean spherical equivalent power, which appears to minimize artifacts even for highly directional micro-structures such as immune cell processes. We highlight examples of positive, negative, and astigmatic power that match expectations according to the known refractive indices and geometries of the relevant structures (for example, a blood vessel filled with plasma acts as a negatively powered cylindrical lens). The examples highlight the benefits of the proposed scheme for the visualization of diverse phase objects including rod and cone inner segments, immune cells near the inner limiting membrane, flowing blood cells, the intravascular cell-free layer, and anatomical details of the vessel wall.