In vivo Imaging of the Human Retinal Pigment Epithelium Cell Mosaic using Short-wavelength Autofluorescence and achromatizing lenses

Abstract

 

Purpose

 

Although adaptive optics (AO) imaging of the human retinal pigment epithelium (RPE) cell mosaic using intrinsic fluorescence has been recently demonstrated, it remains difficult to perform. This is mostly due to light safety limitations, the presence of longitudinal chromatic aberration (LCA) in the eye and more importantly, its variation across individuals. This last point complicates the focusing of the illumination (excitation) and the imaging (emission) channels, as they need to be adjusted for each individual. Here we explore the use of achromatizing lenses to mitigate this problem by compensating for the LCA that would be found in an average eye, thus bringing to closer focus the excitation and emission wavelength ranges.

 

Methods

 

An AO scanning light ophthalmoscope (AOSLO) that allows simultaneous near-infrared (NIR) and visible imaging was modified with achromatizing lenses in the pupil planes of the illumination and imaging paths. Images were recorded in four subjects using a 2° field of view, 30 μW of 850 nm light for wavefront sensing, 92 μW of 790 nm light for reflectance imaging and 60 μW of 560-570 nm light for fluorescence excitation. At each retinal location, the excitation was first brought into focus onto the photoreceptor layer in reflectance, and then shifted towards the RPE. This was followed by simultaneous NIR reflectance and fluorescence imaging during 60-120 seconds using a 2.5 Airy disk confocal aperture and a 625 nm central wavelength (90 nm bandwidth) in the visible channel.

 

Results

 

Imaging the photoreceptor mosaic using a broadband excitation light source (10 nm bandwidth) in reflectance greatly facilitates the focusing of the RPE imaging in fluorescence. The contiguous RPE cell mosaic can be visualized using safe light levels in human subjects after recording images at multiple foci.

 

Conclusions

 

RPE imaging at the cellular scale is facilitated by the use of achromatizing lenses, although further improvement to this method requires accounting for inter-subject variations of LCA. The ability to visualize the RPE mosaic holds promise for studying and diagnosing retinal degenerations such as age related macular degeneration, as well as evaluating new therapies.

 

 

RPE cell mosaic in two different human subjects, with the fluorescent signal originating from lipofuscin containing granules (scale bars are 50 μm across).

 

Keywords: 701 retinal pigment epithelium • 552 imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • 688 retina