Human Retinal Pigment Epithelium: In Vivo Cell Morphometry, Multispectral Autofluorescence, and Relationship to Cone Mosaic

Abstract

PurposeTo characterize in vivo morphometry and multispectral autofluorescence of the retinal pigment epithelial (RPE) cell mosaic and its relationship to cone cell topography across the macula.MethodsRPE cell morphometrics were computed in regularly spaced regions of interest (ROIs) from contiguous short-wavelength autofluorescence (SWAF) and photoreceptor reflectance images collected across the macula in one eye of 10 normal participants (23–65 years) by using adaptive optics scanning light ophthalmoscopy (AOSLO). Infrared autofluorescence (IRAF) images of the RPE were collected with AOSLO in seven normal participants (22–65 years), with participant overlap, and compared to SWAF quantitatively and qualitatively.ResultsRPE cell statistics could be analyzed in 84% of SWAF ROIs. RPE cell density consistently decreased with eccentricity from the fovea (participant mean ± SD: 6026 ± 1590 cells/mm2 at fovea; 4552 ± 1370 cells/mm2 and 3757 ± 1290 cells/mm2 at 3.5 mm temporally and nasally, respectively). Mean cone-to-RPE cell ratio decreased rapidly from 16.6 at the foveal center to <5 by 1 mm. IRAF revealed cells in six of seven participants, in agreement with SWAF RPE cell size and location. Differences in cell fluorescent structure, contrast, and visibility beneath vasculature were observed between modalities.ConclusionsImprovements in AOSLO autofluorescence imaging permit efficient visualization of RPE cells with safe light exposures, allowing individual characterization of RPE cell morphometry that is variable between participants. The normative dataset and analysis of RPE cell IRAF and SWAF herein are essential for understanding microscopic characteristics of cell fluorescence and may assist in interpreting disease progression in RPE cells.

In vivo near-infrared autofluorescence imaging of retinal pigment epithelial cells with 757 nm excitation

“…For subject #4, the RPE mosaic was clearly resolved from the fovea to 3° temporal. This was followed by a reduced NIRAF signal of low contrast in an annular region around the fovea from 3 to 5°, in agreement with previous findings from SWAF [24]. Interestingly, this area contained a few very bright hyper-AF cells ( Fig.…”

“…We typically acquired 800 frames at 24 fps, requiring 30 seconds to record each movie. In comparison to other NIRAF AO imaging [16,[23][24][25], our excitation wavelength of 757 nm should give higher melanin absorption compared to the longer wavelength excitation light near 790 nm that they used. We also used a shorter wavelength emission band compared to previous studies, 778 nm to 810 nm versus 800 nm to 830 nm in [16,[23][24][25].…”

“…In comparison to other NIRAF AO imaging [16,[23][24][25], our excitation wavelength of 757 nm should give higher melanin absorption compared to the longer wavelength excitation light near 790 nm that they used. We also used a shorter wavelength emission band compared to previous studies, 778 nm to 810 nm versus 800 nm to 830 nm in [16,[23][24][25]. Light levels were 0.75 mW for the 2° long line scan 840 nm beacon and 0.9 mW for the 2° × 2° field 757 nm AOSLO beam, whose overlapping combination, when considering the fraction of the ANSI maximum permissible exposure (MPE) of each source, represents 80% of the total MPE as defined by ANSI [28].…”

“…Adaptive optics scanning laser ophthalmoscopy (AOSLO) [20] has been used to image SWAF [21,22] and NIRAF [16,23,24] in healthy volunteers and reveals the individual cells of the RPE mosaic in vivo. NIRAF has also been used to image the RPE mosaic after administration of ICG [25].…”

Longitudinal adaptive optics fluorescence microscopy reveals cellular mosaicism in patients

“…AO-ICG does not cause any detectable bleaching of the ICG fluorescence (Supplemental Figure 7 and Supplemental Table 5, P = 0.45, 1-tailed paired t test); it should be noted that the light levels used here, 135 μW of 790 nm, are less than those reported to cause bleaching (160 μW of 796 nm light; ref. 38), but there could be other factors, as well. Although administration of ICG is not without risks, the risk for adverse reactions is very low (39), and there is potentially information about the status of RPE cells that can only be gleaned from a technique such as AO-ICG.…”