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O. La Schiazza, M. Agopov, M. Han, J. Bille; Comparison of One-Photon and Two-Photon Excited Autofluorescence of RPE Cells From Human Donor Eyes. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2589.
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Two-photon excited fluorescence (TPEF) on RPE cells offers a number of advantages compared to one-photon excited fluorescence in the human eye, e.g. large sensing depth, minimized photo damage and sub-micron resolution. The present study compares blue excited one-photon and red excited two-photon autofluorescence of RPE cells from human donor eyes in terms of resolution, sensitivity and emitted spectrum.
A DPSS 488 nm (Kyma, Melles Griot) cw- resp. Ti:Sapphire (780 nm, 150 fs, 76 MHz) (Mira 900F, Coherent) fs-laser was coupled to a conventional laser scanning ophthalmoscope (HRT, Heidelberg Engineering GmbH, Heidelberg, Germany). RPE samples from human donor eyes were imaged with both lasers used as excitation source. The one-photon detection channel used a confocal pinhole in front of the photodiode, wereas in the two-photon detection channel intrinsic depth discrimination was achieved without the confocal pinhole. Autofluorescence and reflection were in both cases separated using dichroic beamsplitters combined with appropriate shortpass filters. The spectrum of the autofluorescence was measured using a VIS-NIR spectrometer from OceanOptics.
Resolution and Sensitivity of TPE autofluorescence on RPE samples were comparable to single photon excitated fluorescence, with the advantages of larger sensing depth, minimized photo damage and intrinsic 3D resolution. The autofluorescence spectrum of the blue excited RPE cells ranged from 500-740 nm, with a emission peak at 560 nm.
Two photon retinal imaging allows to study a broad range of pathological and morphological defects in the retina with higher resolution and less photo- and thermal damage out of focus compared to a single photon blue light excitation and has the potential to find a main pillar in clinical ophthalmological applications with the possibility to take this method through to patients with retinal disease, e.g. AMD.
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