In-vivo imaging of retinal nerve fiber layer vasculature: imaging histology comparison.

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. METHODS: We injected sodium fluorescein intravenously in two macaque monkeys while imaging the retina with an FAO-SLO. An argon laser provided the 488 nm excitation source for fluorescence imaging. Reflectance images, obtained simultaneously with near infrared light, permitted precise surface registration of individual frames of the fluorescence imaging. In-vivo imaging was then compared to ex-vivo confocal microscopy of the same tissue. RESULTS: Superficial focus (innermost retina) at all depths within the NFL revealed a vasculature with extremely long capillaries, thin walls, little variation in caliber and parallel-linked structure oriented parallel to the NFL axons, typical of the radial peripapillary capillaries (RPCs). However, at a deeper focus beneath the NFL, (toward outer retina) the polygonal pattern typical of the ganglion cell layer (inner) and outer retinal vasculature was seen. These distinguishing patterns were also seen on histological examination of the same retinas. Furthermore, the thickness of the RPC beds and the caliber of individual RPCs determined by imaging closely matched that measured in histological sections. CONCLUSION: This robust method demonstrates in-vivo, high-resolution, confocal imaging of the vasculature through the full thickness of the NFL in the living macaque, in precise agreement with histology. FAO provides a new tool to examine possible primary or secondary role of the nerve fiber layer vasculature in retinal vascular disorders and other eye diseases, such as glaucoma.

In vivo imaging of the fine structure of rhodamine-labeled macaque retinal ganglion cells.

PURPOSE: The extent to which the fine structure of single ganglion cells, such as dendrites and axons, can be resolved in retinal images obtained from the living primate eye was investigated. METHODS: Macaque retinal ganglion cells were labeled with retrograde transport of rhodamine dextran injected into the lateral geniculate nucleus. Fluorescence images of the ganglion cells were obtained in vivo with an adaptive optics scanning laser ophthalmoscope. RESULTS: Axons and dendritic arborization could be resolved in primate retinal ganglion cells in vivo, comparing favorably in detail with ex vivo confocal images of the same cells. The full width at half maximum of the transverse line spread function (LSF) was 1.6 microm, and that of the axial point spread function (PSF) was 115 microm. The axial positional accuracy of fluorescence-labeled objects was approximately 4 microm. CONCLUSIONS: 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.

In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells.

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.