The ability to resolve single cells noninvasively in the living retina has important applications for the study of normal retina, diseased retina, and the efficacy of therapies for retinal disease. We describe a new instrument for high-resolution, in vivo imaging of the mammalian retina that combines the benefits of confocal detection, adaptive optics, multispectral, and fluorescence imaging. The instrument is capable of imaging single ganglion cells and their axons through retrograde transport in ganglion cells of fluorescent dyes injected into the monkey lateral geniculate nucleus (LGN). In addition, we demonstrate a method involving simultaneous imaging in two spectral bands that allows the integration of very weak signals across many frames despite inter-frame movement of the eye. With this method, we are also able to resolve the smallest retinal capillaries in fluorescein angiography and the mosaic of retinal pigment epithelium (RPE) cells with lipofuscin autofluorescence.

Background: Although it has been suggested that alterations of nerve fiber layer vasculature may be involved in the etiology of eye diseases, including glaucoma, it has not been possible to examine this vasculature in-vivo. This report describes a novel imaging method, fluorescence adaptive optics (FAO) scanning laser ophthalmoscopy (SLO), that makes possible for the first time in-vivo imaging of this vasculature in the living macaque, comparing in-vivo and ex-vivo imaging of this vascular bed.

This in vivo method applied to ganglion cells demonstrates that structures smaller than the somas of typical retinal cells can be accessible in living eyes. Similar approaches may be applied to image other relatively transparent retinal structures, providing a potentially valuable tool for microscopic examination of the normal and diseased living retina.

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