Abstract
Purpose :
Zeaxanthin and meso-zeaxanthin are the dominant carotenoids in the human fovea. These two carotenoids are stereoisomers, sharing the same molecular formula but differing from each other only in the orientation of the hydroxyl group at the C3’ of the β-ionone ring. Conventional resonance Raman microscopy cannot distinguish zeaxanthin from meso-zeaxanthin. Here, we investigated whether zeaxanthin and meso-zeaxanthin can be separately measured using polarized resonance Raman microscopy.
Methods :
Our XploRA Plus confocal resonance Raman microscope is equipped with three polarized laser modes (vertical, horizontal, and circular) and three polarized Raman detection modes (vertical, horizontal, and non-polarized), generating nine polarized test conditions. To determine the optimal condition for the separation of zeaxanthin and meso-zeaxanthin, 100 µM zeaxanthin and 100 µM meso-zeaxanthin solutions in methanol were mixed with ratios of 1:0, 3:1, 2:1, 1:1, 1:2, 1:3, and 0:1, respectively, and tested at nine polarized conditions with excitation of a 473 nm laser. Using the optimized condition, we further measured the carotenoid in the fovea of an 87-year-old female donor eye. A total zeaxanthin map was created by mapping the intensity of the V1 peak at 1528 cm-1. Separate maps of zeaxanthin and meso-zeaxanthin were generated by LabSpec6 software’s Classical Least Squares(CLS) fitting algorithm.
Results :
The compositions of zeaxanthin and meso-zeaxanthin in the mixed carotenoid solutions measured under two polarized conditions: horizontal laser vs vertical Raman (H-V), and circular laser vs vertical Raman (C-V), were significantly correlated to the known ratios of the mixed carotenoid solutions. The Raman signals of carotenoids under C-V condition were stronger than the H-V condition, serving as the optimized condition to measure carotenoids in the human fovea. The retinal distribution of total zeaxanthins exhibits a circle with a dot at its center. Further analysis revealed that zeaxanthin was localized mainly at the central dot area, whereas the meso-zeaxanthin was allocated on the circle.
Conclusions :
This demonstrates that zeaxanthin and meso-zeaxanthin can be separately measured under polarized Raman conditions, which might offer new knowledge to understand the physiological role of macular carotenoids in the prevention of retinal disease.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.