Abstract
Purpose :
The retinal pigment epithelium (RPE) plays an essential role in the physiology of the eye by nourishing photoreceptors and maintaining retinal integrity. Its dysfunction is associated with diseases such as age-related macular degeneration and retinitis pigmentosa. Clinically, autofluorescence for lipofuscin at short-wavelength excitation (488 nm) and melanin at near-infrared excitation (785 nm) has been used to evaluate RPEs. Both are known to change during diseases. Here we show a combined approach to assess both fluorophores simultaneously.
Methods :
We converted a two-photon adaptive optics scanning laser ophthalmoscope (AOSLO) into a microscope (objective: Nikon 16X, 0.80 NA) to perform single-photon, two-photon and dark-field imaging simultaneously at an excitation wavelength of 730 nm using a pulsed laser. Melanin was targeted via single-photon excitation and lipofuscin via two-photon excitation. Fresh ex-vivo porcine RPE samples were imaged. Excitation power of ~3 mW was used for 60 s exposures over 150 µm x 125 µm field. Fluorophores distribution and cell regularity was assessed for all images.
Results :
Continuous mosaics of individual RPE cells were detected in all channels, however, with distinct features. The regularity analysis showed a mean cell size of 10.85 ± 0.55 μm. In the dark-field channel, the morphology of RPE cells were revealed by cell membranes appearing bright and subcellular features (e.g. apical microvilli). In single photon fluorescence, autofluorescence was limited to the cytosol, occasionally showing hypofluorescent structures. In two-photon fluorescence, individual cells appeared more defined in some samples, hyperfluorescent structures were visible that accumulated mostly along the cell membranes.
Conclusions :
We have shown the feasibility of our modified AOSLO setup for simultaneous single-photon and two-photon imaging to assess RPE autofluorescence. The approach limits the exposure delivered to the tissue and minimises uncertainties regarding focal depth and changes over time. With further improvements in efficiency this form of multimodal autofluorescence imaging might be applied in clinical diagnostics for humans.
This abstract was presented at the 2024 ARVO Imaging in the Eye Conference, held in Seattle, WA, May 4, 2024.