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

Abstract

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.

Imaging light responses of retinal ganglion cells in the living mouse eye

“…It utilized an AO imaging system (Geng et al 2012;Gray et al 2006Gray et al , 2008 to obtain highresolution images of fluorescing cells in retina despite the challenges of low fluorescence levels and eye movements. We employed an UV light source that activates the S-cone opsin to measure ganglion cell responses in the presence of the blue imaging laser, which also excites M-cone opsin and rhodopsin.…”

Imaging retinal mosaics in the living eye

“…Fluorescence adaptive optics scanning laser ophthalmoscopy (FAOSLO) overcomes the resolution limitations of current clinical instruments allowing individual RPE cells to be imaged in living macaque ( Figure 3a) and human eyes. 21,22 The exclusion of lipofuscin from the nuclei of RPE cells and the fact that it is sequestered in granules in the cell cytoplasm generates contrast in images of single cells. 23,22 FAOSLO uses two different sources that can be used for simultaneous high signal-to-noise ratio (SNR) reflectance imaging with low SNR fluorescence imaging.…”

“…21,22 The exclusion of lipofuscin from the nuclei of RPE cells and the fact that it is sequestered in granules in the cell cytoplasm generates contrast in images of single cells. 23,22 FAOSLO uses two different sources that can be used for simultaneous high signal-to-noise ratio (SNR) reflectance imaging with low SNR fluorescence imaging. Reflectance imaging in the infrared can be used to image photoreceptors, whereas autofluorescence imaging with visible light excitation and emission can be used for RPE mosaic imaging.…”

“…Reflectance imaging in the infrared can be used to image photoreceptors, whereas autofluorescence imaging with visible light excitation and emission can be used for RPE mosaic imaging. 23 It has been shown that high resolution RPE autofluorescence imaging in FAOSLO can reliably and repeatedly obtain images of the complete RPE cell mosaic. 22 In normal human retina, images were obtained at the same retinal location at different time points with 100% of the cells able to be identified in both images.…”

“…Gray et al showed that individual RGC bodies, axons, and dendrites could be visualized using adaptive optics retinal imaging in living macaque eyes when a fluorescent dye was introduced into RGCs via retrograde transport after injection into the LGN (Figure 6). 21,3 Ganglion cell bodies, axons, and dendrites labeled with eGFP have also been visualized in living rat eyes using FAOSLO. 29 These techniques are too invasive for use in humans, but many of the studies that are particularly relevant for understanding RGC function are perhaps best applied in animal models.…”