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D. Schweitzer, S. Jentsch, S. Schenke, M. Hammer, E. R. Gaillard; Excitation- and Emission Spectra as Well as Auto Fluorescence Lifetime of Anatomical Structures of the Eye. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3793.
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© ARVO (1962-2015); The Authors (2016-present)
Investigation of metabolism at the eye can be performed by measurement of tissue auto fluorescence. Information related to single fluorophores requires the investigation of specific excitation and emission as well as of the fluorescence lifetime. These properties are determined for the main ocular structures of porcine eyes in a basic study.
Anatomical structures of porcine eyes were separated about one hour after scarifying the animals in a slaughterhouse. Parts of cornea, chamber water, lens, vitreous, neuronal retina retinal, pigment epithelium, choroid, and sclera were inserted in cuvettes. The spectra of absorbance, excitation, and emission were measured with spectrometers Lambda 2 UV/VIS and LS 5 (Perkin Elmer). The fluorescence lifetimes were determined exciting the samples by 100 ps pulses at 446 nm, applying the Jena Ophthalmo-lifetime-mapper.
The absorbance increases at shorter wavelengths with local maxima around 200 and 270 nm. In contrast to primate eyes, the absorption edges of cornea and lens are at 300 and 350 nm. Measuring the fluorescence at 460, 530, and 650 nm, maxima in the excitation spectra appear at 225, 300, 340, 385, 440, and 470 nm but different between the ocular structures.Excitation at 300, 360, 400, 446, and 470 nm results in fluorescence maxima at 340, 460, 495, 530, 565, 600, 625 and 650 nm. The bi-exponential approximation of dynamic fluorescence results in further discrimination of ocular tissue by Tau 1 (200, 254, 357, 443, and 538 ps), Tau 2 (2.2, 2.7, 3.2, 3.4, and 3.6 ns), and the amplitude A1 (7 to 37%). Tri-exponential approximation of lipofuscin in an intersection of a human donor results in Tau 1 = 72 ps, A1 = 71%, Tau 2 = 373 ps, A2= 27.2%, and Tau3 = 1661 ps, A3 = 1.8%. Bruch's membrane is bi-exponential approximated (Tau 1 = 690 ps, A1= 69.5%, Tau 2 = 4170 ps, A2 = 30.5%).
Contribution of several endogenous fluorophores can be determined in different parts of porcine eyes. The fluorescence maxima at 460 and 530 nm correspond to NADH and FAD. Connective tissue can clearly be discriminated from other fluorophores by fluorescence lifetime. There is a good correspondence in lifetimes of sclera to collagen II and of Bruch's membrane with collagen I.
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