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Jennifer J Hunter, Robin Sharma, Grazyna Palczewska, Krzysztof Palczewski, David R Williams; In vivo two-photon fluorescence imaging of primate inner retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5970.
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© ARVO (1962-2015); The Authors (2016-present)
Adaptive optics retinal imaging in vivo has enabled imaging of vasculature and nerve fiber bundles in the inner retina, but the contrast from other structures is weak. Most cells contain fluorophores that could provide sufficient contrast, but they cannot be accessed through conventional means. We used two-photon fluorescence (TPF) to image autofluorescent structures in the inner retina of living primates.
A two-photon adaptive optics (AO) scanning light ophthalmoscope was used to repeatedly image TPF in 3 macaques (730 nm excitation, 400-550 nm emission). TPF signals from inner retinal layers were recorded for up to 10 minutes and then averaged. Near infrared reflectance images were simultaneously acquired for dual registration. AO was used to focus through different layers in the retina. For histological comparison, TPF microscopy was conducted in fixed, flat-mounted retina in one of these macaques.
When focused at the nerve fiber layer, a weak TPF was captured from fiber bundles. Greater TPF was observed from streak-like structures that were co-localized with dark gaps between the axon bundles in reflectance. These streaks could represent Müller cell processes known to occupy the spaces between nerve fiber bundles, although the fluorophores remain unknown.<br /> <br /> In a narrow focus window just beneath these streaks, a mosaic of dark, circular features was observed. This mosaic resembles ex vivo images of the ganglion cell layer. Analysis of Fourier spectra revealed local maxima at similar spatial frequencies for the in vivo and ex vivo images from the same retinal eccentricities implying that the density of the dark features is the same as that of ganglion cell bodies.<br /> <br /> TPF also emanated from within the walls of vessels in the inner retina. When the beam was focused on the superficial vessel walls, TPF from the surface was captured. For deeper focus values, TPF from a cross-section of the vessel walls appeared as two bright bands. Larger vessels near the optic disk were more well-defined than smaller ones away from the disk. Fluorescence from vessel walls is likely due to proteins such as collagen and elastin.
We have demonstrated non-invasive two-photon imaging of structures within the inner retina of the living primate eye. This visualization of otherwise unseen morphological features in the inner retina could be a useful tool to investigate their structural integrity in normal and diseased eyes.
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