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M. Sharifzadeh, D.Y. Zhao, P.S. Bernstein, W. Gellermann; In vitro Raman Imaging of Macular Pigments in Human Donor Eyes . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1779.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have used resonance Raman spectroscopy (RRS) in an imaging mode to investigate macular pigment (MP) distributions in human donor eyes. The purpose is to use RRS imaging as a direct, rapid, two–dimensional optical technique to measure MP levels as well as to obtain highly molecule–specific information about the spatial distribution of MP in the human macula. Methods: We measured more than 40 donor eyes having wide age variation (24 to 90 years). All samples were free of macular and retinal diseases. Using a narrow– band tunable filter, 488 nm argon laser excitation, a suitably designed light delivery and collection module, and detection with a CCD camera, we were able to record RRS images of the macular region that originate from the C=C double–bond stretch vibrations of the MP molecules. To generate an image, we recorded two sets of pixel maps. First, an on– peak map, containing Raman and fluorescence background signals, and second, an off– peak map, containing only the fluorescence background. The two maps were then digitally subtracted. Following optical MP detection, the concentrations of MP were measured using the traditional gold standard of HPLC. Results: We obtained excellent correlation between MP levels obtained by RRS imaging and HPLC (R=0.92, P<0.0001). Taking advantage of the high chemical specificity of RRS spectroscopy and the imaging modality, we were able to obtain information about the MP distributions in the human macula with high spatial resolution. The images reveal several distinct classes of possible pigment distributions; the correlation results validate the non–invasive Raman detection method. Conclusions: Excellent correlation between RRS imaging and HPLC confirms that Raman imaging is a non–invasive, reliable optical technique yielding information regarding concentration levels and detailed spatial distributions of macular pigment. The technique therefore holds promise for in–vivo applications.
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