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Binxing Li, Evan George, Gregory T. Rognon, Fu-Yen Chang, Jeanne M Frederick, Jonathan Stoddard, Trevor J McGill, Martha Neuringer, Paul S Bernstein; Imaging Lutein and Zeaxanthin in the Primate Macula by Confocal Resonance Raman Microscopy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5840.
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Supplementation with lutein and/or zeaxanthin can reduce the risk of human eye disease such as age-related macular degeneration (AMD) and improve visual function. However, until now, the differential distributions of lutein and zeaxanthin remain undetermined due to lack of instrumentation which could distinguish between these carotenoids in situ. Using an advanced confocal resonance Raman microscope, we investigated the distribution of lutein and zeaxanthin in the primate macula.
We first tested if the spectrometer of the confocal resonance Raman microscope can distinguish lutein from zeaxanthin by measuring the Raman spectra of lutein and zeaxanthin in methanol using gratings of 600, 1200, 1800, and 2400 gr/mm. Using optimized parameters, we then mapped the 3-D distribution of lutein and zeaxanthin in an 8-mm macular punch from a formalin fixed eyeball of a 77-year-old female donor. We also examined cross-sections of human and monkey fovea. All the experiments were carried out on a Horiba XploRA PLUS confocal resonance Raman microscope at 0 °C. An Olympus BX41 confocal microscope with 10X objective was coupled to the Raman spectrometer, and the excitation light was provided by a 473-nm laser. Data were obtained and analyzed with Horiba’s LabSpec 6 software.
Among the four Raman gratings, the 1800 gr/mm grating was the best. The Raman shifts of the peaks of the C=C bond vibrations of lutein and zeaxanthin were distinguishable at 1532 cm-1 and 1528 cm-1, respectively, without significantly losing intensity. On the macular punch, both lutein and zeaxanthin were concentrated at the fovea in a region about 0.5 mm diameter. A 300-µm-deep scan revealed that lutein is present dominantly in the outer plexiform layers (OPL) near the bipolar cells, while zeaxanthin becomes the major component with increasing depth. Cross-section Raman images of carotenoid in the primate fovea produced images comparable to the classic Snodderly blue light absorbance images published in 1984.
Confocal resonance Raman microscopy enables us to distinguish lutein from zeaxanthin. Our findings of the specific spatial distribution of lutein and zeaxanthin in the human retina may facilitate our understanding on the function of the macular carotenoids.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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