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O. La Schiazza, J. F. Bille; Fast Two-Photon Excited Autofluorescence Imaging of the Neurosensory Retina and Underlying RPE Cells from Human Donor Eyes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1844. doi: https://doi.org/.
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Two-photon excited fluorescence imaging offers a number of advantages compared to convential single-photon excited fluorescence screening methods, e.g. large sensing depth, minimized photo damage and intrinsic 3D-resolution without the need of a confocal pinhole. The present study focuses on the implementation of two-photon microscopy combined to a fast resonant ophthalmologic scanner for a possible application in the human eye for retinal disease diagnostic.
A Ti:Sapphire (830 nm, 150 fs, 76 MHz) (Mira 900F, Coherent) fs-laser was coupled to a conventional laser scanning ophthalmoscope (Heidelberg Retina Tomograph HRT). The neurosensory retina and underlying RPE cells from human donor eyes were imaged in two-photon exited autofluorescence. The autofluorescence signal is separated from backscattered light using a dichroic beamsplitter combined with appropriate shortpass filters.
Two-photon excited autofluorescence signals could be detected from the photoreceptors outer as well as inner segments. Furthermore the retinal nerve fiber layer and the underlying ganglion cells could be imaged using two-photon excited autofluorescence. Accumulated lipofuscin in the RPE cells shows a strong autofluorescent signal that could be excited and detected through the super-imposed neurosensory retina when excited with NIR fs-laser light.
Two-photon imaging of intrinsic retinal fluorophores allows to study a broad range of pathological and morphological defects in the retina with higher resolution and less photo- and thermal out-of-focus damage compared to a single-photon blue light excitation. The neurosensensory retina contains autofluorescent markers that enable direct visualization of retinal health and can be used for diagnostic and monitoring of retinal diseases. Excitation of the RPE cells through the neurosensory retina by NIR laser light, generates enough fluorescence signal for a quantitative estimation of RPE’s lipofuscin content.
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