Origin of cell contrast in offset aperture adaptive optics ophthalmoscopy.

Offset aperture and split detector imaging are variants of adaptive optics scanning ophthalmoscopy recently introduced to improve the image contrast of retinal cells. Unlike conventional confocal scanning ophthalmoscopy, these approaches collect light laterally decentered from the optical axis. A complete explanation of how these methods enhance contrast has not been described. Here, we provide an optical model with supporting in vivo data that show contrast is generated from spatial variations in the refractive index as it is in phase contrast microscopy. A prediction of this model is supported by experimental data that show contrast is optimized when the detector is placed conjugate with a deeper backscattering screen such as the retinal pigment epithelium and choroid, rather than with the layer being imaged as in conventional confocal imaging. This detection strategy provides a substantial improvement in the contrast these new methods can produce.

Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye.

Measuring blood cell dynamics within the capillaries of the living eye provides crucial information regarding the health of the microvascular network. To date, the study of single blood cell movement in this network has been obscured by optical aberrations, hindered by weak optical contrast, and often required injection of exogenous fluorescent dyes to perform measurements. Here we present a new strategy to non-invasively image single blood cells in the living mouse eye without contrast agents. Eye aberrations were corrected with an adaptive optics camera coupled with a fast, 15 kHz scanned beam orthogonal to a capillary of interest. Blood cells were imaged as they flowed past a near infrared imaging beam to which the eye is relatively insensitive. Optical contrast of cells was optimized using differential scatter of blood cells in the split-detector imaging configuration. Combined, these strategies provide label-free, non-invasive imaging of blood cells in the retina as they travel in single file in capillaries, enabling determination of cell flux, morphology, class, velocity, and rheology at the single cell level.

Imaging translucent cell bodies in the living mouse retina without contrast agents.

The transparency of most retinal cell classes typically precludes imaging them in the living eye; unless invasive methods are used that deploy extrinsic contrast agents. Using an adaptive optics scanning light ophthalmoscope (AOSLO) and capitalizing on the large numerical aperture of the mouse eye, we enhanced the contrast from otherwise transparent cells by subtracting the left from the right half of the light distribution in the detector plane. With this approach, it is possible to image the distal processes of photoreceptors, their more proximal cell bodies and the mosaic of horizontal cells in the living mouse retina.