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L. Zhao, J. Qu, G. Xu, Y. Sun, D. Chen, H. Niu; Fluorescence Spectroscopy of Retinal Pigment Epithelium Cells: Single- and Two-Photon Excitation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2620.
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Previous research in histology and spectroscopy shows that RPE cells are abundant in endogenous fluorophores, such as lipofuscin, FAD, NADH etc. The purpose of this work is to investigate the components, distributions and spectra of these fluorophores, which potentially provides clues for the study of pathological mechanisms and early clinical diagnosis of the ocular fundus diseases.
Fresh porcine eyes are harvested within 4 hours after death. A 6mm-diameter RPE-choroid-sclera complex near the macula is trephined and placed into a specially designed chamber with a coverslip. Fluorescence spectroscopy of the sample is performed on an inverted laser scanning confocal microscope. An Argon ion laser and a mode-locked femtosecond Ti:sappire laser are coupled to the input ports of the microscope for single- and two-photon excitation of the sample. The excitation wavelengths for single- and two-photon excitation in this study are 458nm and 860nm, respectively.
Fluorescence spectroscopy is performed to the same region of the sample, and two-photon excitation measurement is made before single-photon excitation. When the detecting wavelength is from 426 to 706 nm, the two-photon excitation fluorescence spectrum has two peaks; one is at 590nm, and the other minor weak at 500nm. The FWHM of the spectrum is about 150nm. Basically, single-photon excitation fluorescence spectrum is consistent with that of two-photon excitation, but has reduced fluorescence intensity. Wide absorption spectrum of two-photon excitation results in the excitation of more fluorophores than single-photon excitation and broader two-photon excitation fluorescence spectrum than that of the single-photon excitation. The main peak of the single- and two-photon excitation spectra(590nm) is corresponding to fluorophore A2E, as reported in the literatures. The minor peak at 500nm is believed to be from Vitamin A.
Two-photon excitation facilitates the simultaneous excitation of different fluorophores in RPE cells and the acquisition of their compound spectra in only one measurement. When combined with other fluorescence contrast measurement, such as fluorescence lifetime, single- and two-photon excitation fluorescence spectroscopy can provide better differentiation of the fluorophores in RPE cells as well as their microenvironment such as pH, oxygen concentration etc. which is key to the monitoring of the metabolic state of RPE cells.
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