Safe real-time imaging of human retinal pigment epithelial cells in the living eye

Abstract

Purpose: Although it has been shown that RPE cell can be imaged with fluorescence AOSLO (FAOSLO) in normal humans [1] and in AMD [2], high light levels, focusing challenges, and post-processing time have limited clinical application of these methods. We have improved FAOSLO to permit routine imaging of the RPE mosaic and montaging of multiple RPE images in a clinical setting.

Methods: We improved RPE imaging efficiency by precisely controlling light exposure with a shutter, restricting visible light delivery to the retina only during data acquisition. This improvement was implemented in a FAOSLO with optical eye tracking and digital image registration, where a fast tip/tilt mirror compensates for eye motion, allowing precise temporal control of the fluorescence excitation light. Real-time co-registration and integration of the fluorescence images, using eye motion signals derived from simultaneously acquired near infrared reflectance channel, allows the RPE image to be visualized immediately. A high-fidelity algorithm runs in real-time to filter out motion artifacts from images after optical eye tracking and digital registration. We demonstrate these methods in both normal and diseased eyes, including AMD and Stargardt’s eyes.

Results: Focus and detector optimization can be accomplished with <10 seconds of total cumulative light exposure, a reduction in total time of ~85%. This improvement was implemented using light levels that were ~40% lower than we used previously. This savings in light budget allowed for multiple RPE images to be obtained from adjacent retinal areas with sufficient overlap to produce RPE image montages. In normal eyes a contiguous mosaic was visible and in diseased eyes RPE mosaic disruption was visible. Real-time eye signal integration of the auto-fluorescence channel allowed RPE images to be displayed to the experimenter instantaneously.

Conclusions: Efficient control of visible light exposure coupled with real-time fluorescence registration and signal integration allows immediate examination of the RPE mosaic on a microscopic scale across large retinal areas in the living eye. These techniques could have broad application in high-resolution retinal imaging.<br /> <br /> [1] Morgan, et. al., Invest. Ophthalmol. Vis. Sci. 50(3), 1350-1359 (2008).<br /> <br /> [2] Rossi, et. al., Biomed. Opt. Express, 4(11), 2527-2539 (2013).<br />