Daniel C. Gray

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.

Light-Induced Retinal Changes Observed with High-Resolution Autofluorescence Imaging of the Retinal Pigment Epithelium

PURPOSE Autofluorescence fundus imaging using an adaptive optics scanning laser ophthalmoscope (AOSLO) allows for imaging of individual retinal pigment epithelial (RPE) cells in vivo. In this study, the potential of retinal damage was investigated by using radiant exposure levels that are 2 to 150 times those used for routine imaging. METHODS Macaque retinas were imaged in vivo with a fluorescence AOSLO. The retina was exposed to 568- or 830-nm light for 15 minutes at various intensities over a square (1/2) degrees per side. Pre- and immediate postexposure images of the photoreceptors and RPE cells were taken over a 2 degrees field. Long-term AOSLO imaging was performed intermittently from 5 to 165 days after exposure. Exposures delivered over a uniform field were also investigated. RESULTS Exposures to 568-nm light caused an immediate decrease in autofluorescence of RPE cells. Follow-up imaging revealed either full recovery of autofluorescence or long-term damage in the RPE cells at the exposure. The outcomes of AOSLO exposures and uniform field exposures of equal average power were not significantly different. No effects from 830-nm exposures were observed. CONCLUSIONS The study revealed a novel change in RPE autofluorescence induced by 568-nm light exposure. Retinal damage occurred as a direct result of total average power, independent of the light-delivery METHOD Because the exposures were near or below permissible levels in laser safety standards, these results suggest that caution should be used with exposure of the retina to visible light and that the safety standards should be re-evaluated for these exposure conditions.

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 imaging of retinal nerve fiber layer vasculature: imaging - histology comparison

BackgroundAlthough 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.MethodsWe 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.ResultsSuperficial 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.ConclusionThis 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 Microscopic Structures in the Rat Retina

PURPOSE The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. The authors have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo. METHODS Enhanced green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague-Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy. RESULTS Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics. Adaptive optics correction reduced the total root mean square wavefront error, on average, from 0.30 microm to 0.05 microm (measured at 904 nm, 1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was approximately 1.84 microm, approximately 82% greater than the FWHM of the diffraction-limited LSF. CONCLUSIONS With perfect aberration compensation, the in vivo resolution in the rat eye could be approximately 2x greater than that in the human eye because of its large numerical aperture (approximately 0.43). Although the fAOSLO corrects a substantial fraction of the rat eyes aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using adaptive optics to investigate the rodent eye in vivo with high resolution.

Recent Advances in Retinal Imaging With Adaptive Optics

Since its first application to retinal imaging nearly a decade ago, adaptive optics has helped researchers make fundamental advances in the understanding of how the human visual system works.

MEMS in adaptive optics scanning laser ophthalmoscopy: achievements and challenges

This work briefly reviews the achievements of adaptive optics scanning laser ophthalmoscopy to date. Then, an instrument designed for testing phase imaging modalities is described, and finally, the requirements for MEMS devices in scanning ophthalmic devices are discussed.

Dual-Wavelength Focusing and Simultaneous Image Registration for In Vivo High-Resolution Retinal Imaging

We describe dual-wavelength, simultaneous retinal imaging with compensation for eye movements and monochromatic and chromatic aberrations. Using lipofuscin autofluorescence, we can resolve human retinal pigment epithelial cells in vivo.

In Vivo High-Resolution Fluorescence Retinal Imaging with Adaptive Optics

We describe a new instrument combining adaptive optics ophthalmoscopy and fluorescence imaging. The instrument is capable of imaging retrograde labeled ganglion cells, intrinsic fluorescence from retinal pigment epithelial cells, and intravenous fluorescein injections in vivo.