In vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic.

The retinal pigment epithelium (RPE) is a monolayer of cells directly behind the photoreceptors that has several functions including participation in the regeneration of retina in the visual cycle and the phagocytosis of photoreceptor outer segments.1 Healthy RPE cells are critical for maintaining the normal structure of the retina and for preserving normal photoreceptor function. Indeed, abnormal RPE cells contribute to disease mechanism and progression in numerous retinal diseases, including age-related macular degeneration, Stargardt’s dystrophy, Best’s disease, and others.1 Despite the important role of the RPE in retinal health, little is known about the RPE cell mosaic structure, mainly because histologic studies on cell density and packing disagree.2–7 In addition, drug and molecular treatments for retinal diseases are advancing8 such that in vivo metrics must be developed to assess disease progression and treatment efficacy on a cellular scale. The present study addresses these points by providing in vivo images of the normal human RPE cells and by quantifying the spatial arrangement of the RPE mosaic. There has been considerable effort recently to develop in vivo retinal imaging modalities with cellular resolution. The use of adaptive optics (AO), which involves measuring the higher-order optical aberrations in the eye with a wavefront sensor and correcting these aberrations with a wavefront corrector, has allowed improvements in contrast and resolution such that it has become possible to routinely characterize the photoreceptor mosaic in both normal and diseased eyes in vivo.9–16 In addition, the combination of AO with a scanning laser ophthalmoscope (SLO) has allowed for the visualization of individual RPE cells in reflectance imaging in retinal locations where the photoreceptor mosaic is missing in cone–rod dystrophy.17 In vivo reflectance imaging of the RPE cells in normal retina has not been possible with AOSLO techniques, because the axial resolution in combination with the unique waveguiding nature of the overlying photoreceptors obscures signal from the RPE. However, the RPE cells are unique in that they accumulate lipofuscin granules in their cytoplasm through the processes of the visual cycle and phagocytosis.18,19 Lipofuscin naturally consists of several autofluorescence molecules,20–22 and therefore the lipofuscin granules provide an autofluorescence signal, unique to the RPE layer, that allows the RPE layer to be imaged.23 In vivo studies of retinal autofluorescence23–31 have shown that the excitation and the emission spectra of lipofuscin are broad,25 that the RPE autofluorescence signal varies over retinal eccentricity,28 and that the signal increases as a function of age until the eighth decade of life, after which the signal tends to decrease.28,32 The lipofuscin autofluorescence signal has been used to image macroscopic features of the RPE layer in both normal and diseased retinas.23,26,27,29–31 This study presents the first in vivo images of the complete RPE cell mosaic in normal human retinas by using a combination of lipofuscin autofluorescence, AOSLO, and simultaneous dual-wavelength imaging and registration. We have previously demonstrated RPE cell imaging capabilities in the primate retina.33 In the present study, we validated the technique by comparing in vivo images of the macaque RPE mosaic to ex vivo images at the same retinal location. In addition, the spatial arrangement of the RPE mosaic was quantified in normal human and macaque retinas. The ability to image the complete RPE cell mosaic noninvasively in vivo allows morphologic studies of normal and diseased RPE cells and will provide a method of tracking disease progression and treatment efficacy in patients and animal models of disease.