Imaging lutein and zeaxanthin in the human retina with confocal resonance Raman microscopy Binxing Lia, Evan W. Georgea, Gregory T. Rognona, Aruna Gorusupudia, Arunkumar Ranganathana, Fu-Yen Changa, Linjia Shia, Jeanne M. Fredericka, and Paul S. Bernsteina,1 aDepartment of Ophthalmology and Visual Sciences, Moran Eye Center, University of Utah School of Medicine, Salt Lake City, UT 84132 Edited by Janet R. Sparrow, Columbia University Medical Center, New York, NY, and accepted by Editorial Board Member Jeremy Nathans April 1, 2020 (received for review December 30, 2019) Lutein and zeaxanthin are xanthophyll carotenoids that are highly developed countries and that carotenoid supplementation can concentrated in the human macula, where they protect the eye reduce the risk of AMD and improve visual performance (25–33). from oxidative damage and improve visual performance. Distin- We and others have long had an interest in understanding how guishing lutein from zeaxanthin in images of the human retina and why the primate macula goes to such great lengths to spe- in vivo or in donor eye tissues has been challenging because cifically concentrate lutein, zeaxanthin, and meso-zeaxanthin at no available technology has been able to reliably differentiate the fovea. While it is possible to distinguish the differential dis- between these two carotenoids, which differ only in the position tributions of lutein and the zeaxanthins on a macroscopic level by = of one C C bond. Here, we report the differential distributions of dissecting tissues, extracting, and analyzing by HPLC, such lutein and zeaxanthin in human donor retinas mapped with con- methods cannot provide microscopic resolution because tissue focal resonance Raman microscopy. Zeaxanthin is highly concen- punches typically have to be several millimeters in diameter. trated in the fovea, extending from the inner to the outer limiting Snodderly’s blue light microscopic method on tissue sections fails membranes, with especially high concentrations in the outer plex- iform layer, while lutein is much more diffuse at relatively lower to distinguish lutein from the zeaxanthins because their visible concentration. Our results imply that zeaxanthin may play a more absorption spectra overlap too much, and other yellow chromo- important role than lutein in human macular health and disease. phores may be present (17, 18). Other in situ methods such as fluorescence microscopy and imaging mass spectrometry likewise MEDICAL SCIENCES lutein | zeaxanthin | Raman | retina | macula fail because carotenoids exhibit only weak fluorescence and could be easily destroyed by the conditions required for in situ mass he macula lutea is an oval-shaped yellow region unique to the spectrometry (34). On the other hand, the MP carotenoids exhibit Tprimate retina, which is responsible for sharp and detailed strong, resonance-enhanced Raman spectra when excited with central vision (1–4). George Wald first recognized in 1945 that blue laser light even in complex tissue matrices (Fig. 1), and due to the yellow color of the macula originated from the deposition of differences in carbon–carbon double-bond conjugation (10 for xanthophyll carotenoids, presumably derived from the diet be- lutein and 11 for zeaxanthin and meso-zeaxanthin), they have cause animals cannot synthesize carotenoids (5). These xantho- subtly different but distinguishable Raman spectra (35, 36). In this phyll carotenoids were chemically identified to be lutein and study, we first optimized the conditions to distinguish lutein from zeaxanthin by Bone and Landrum in 1985, and a few years later, the zeaxanthins using a high-resolution confocal resonance Raman they determined that lutein was exclusively in the 3R,3′R,6′R microscope. Using these optimized methods, we separately mea- configuration commonly found in plants and microorganisms, sured the distributions of lutein and the zeaxanthins in human while zeaxanthin was a nearly equal mixture of dietary 3R,3′R- retinal sections and in Z-stack images of a flat-mounted human zeaxanthin and nondietary 3R,3′S-meso-zeaxanthin, with a very retina. small amount of the nondietary 3S,3′S isomer (6, 7). The P.S.B. laboratory and coworkers found that ∼20% of the macular pigment Significance (MP) carotenoids are oxidation products of lutein and zeaxanthin and that meso-zeaxanthin is produced from lutein by the RPE65 We have determined the spatial distribution of the macular enzyme in the retinal pigment epithelium (RPE) (8–11). In the pigment carotenoids lutein and zeaxanthin in the human retina peripheral retina, the ratio of lutein:zeaxanthin:meso-zeaxanthin is using confocal resonance Raman microscopy and found that about 3:1:0 when measured by high-performance liquid chroma- zeaxanthin is highly concentrated in the fovea, while lutein is tography (HPLC), while in the macula lutea, the concentration of more diffusely spread across the macula at a relatively lower total carotenoids rises 100-fold, and the ratio changes to 1:1:1 concentration. Our results imply that zeaxanthin may play a (12–16). more important role than lutein in human macular health and Snodderly studied the cross-sectional localization of the MP disease, and they demonstrate the elegant ability of confocal carotenoids in 1984 in sections of monkey retina using blue-light resonance Raman imaging to probe the biochemistry and microscopy. At the fovea, the majority of the carotenoids were structure of the most important region of the human retina. present in the outer plexiform layer and the inner plexiform layer, 2 of the 10 layers of the primate retina (17, 18). This focal Author contributions: B.L. and P.S.B. designed research; B.L., E.W.G., G.T.R., A.G., A.R., F.-Y.C., L.S., and J.M.F. performed research; B.L., A.G., A.R., J.M.F., and P.S.B. analyzed distribution of macular carotenoids has been ascribed to the data; and B.L. and P.S.B. wrote the paper. presence of specific binding proteins for the zeaxanthins (GSTP1) The authors declare no competing interest. – and lutein (StARD3) in the human macula (19 21). Besides being This article is a PNAS Direct Submission. J.R.S. is a guest editor invited by the able to absorb short-wavelength blue light (1, 2, 4, 22), lutein and Editorial Board. zeaxanthin are well-known natural antioxidants that can quench Published under the PNAS license. free radicals and singlet oxygen (23, 24). Furthermore, clinical 1To whom correspondence may be addressed. Email: [email protected]. studies have demonstrated that high macular pigment optical This article contains supporting information online at https://www.pnas.org/lookup/suppl/ density (MPOD) is inversely associated with the risk of age-related doi:10.1073/pnas.1922793117/-/DCSupplemental. macular degeneration (AMD), a common cause for blindness in www.pnas.org/cgi/doi/10.1073/pnas.1922793117 PNAS Latest Articles | 1of7 Downloaded by guest on September 26, 2021 relative to these carotenoids dissolved in detergent (37). Lutein, zeaxanthin, lutein-StARD3 protein complexes, and zeaxanthin- GSTP1 protein complexes were dissolved in phosphate buffered saline (PBS) buffer with 8 mM 3-[(3-Cholamidopropyl) dimethy- lammonio]-1-propanesulfonate hydrate (CHAPS), a zwitterionic detergent used to increase the aqueous solubility of carotenoids and carotenoid–protein complexes, and their Raman spectra were then measured. SI Appendix,Fig.S2demonstrates that the overall spectra are unchanged, and the v1 peaks of lutein and zeaxanthin − are still at 1,532 and 1,528 cm 1, respectively, whether dissolved in detergent alone or bound to protein, although the signal intensity becomes weaker when they are bound to protein. Resonance Raman Imaging of Macular Carotenoids in Tissue Sections. Using the optimized Raman methods, we first mapped the total carotenoids in a retinal section from a healthy donor eye with excitation from a 473-nm blue laser. The intensity map of total carotenoids was created using the peak intensity of each Raman Fig. 1. Chemical structures and a typical Raman spectrum of macular ca- −1 rotenoids. (A) Lutein. (B) Zeaxanthin. (C) A Raman spectrum of zeaxanthin, spectra of the v1 peak between 1,500 and 1,550 cm in a region demonstrating the typical Raman spectrum of a carotenoid. Three major 1,800 μm × 500 μm centered at the foveal pit. Fig. 4 shows that − peaks position at about 1,500 (v1), 1,150 (v2), and 1,000 cm 1 (v3). They most carotenoids localize to the 700-μm-wide foveal depression originate from vibrations of conjugated C = C double bonds, C-C single with a steep drop to barely detectable levels more peripherally. bonds, and methyl groups in the carotenoid molecules, respectively. In Cross-sectionally at the foveal center, the strongest Raman sig- theory, a carotenoid with longer C = C double-bond conjugation positions its nals were detected in the OPL (also known as Henle’s fiber v1 peak at a smaller Raman frequency, making it possible to distinguish layer) and the ONL. lutein from zeaxanthin. Fig. 5 shows the Raman spectra at various selected points on the Raman intensity map of Fig. 4. All three spots in the inner retina exhibit a typical carotenoid spectrum, with the spot at the Results center showing the strongest Raman intensity and progressively Optimization of Raman Signal Acquisition. The total macular ca- weaker signals with increasing distance from the fovea. The fo- rotenoid concentration is about 1 mM at the fovea and drops to veal ONL also manifests a strong and clear Raman spectrum less than 10 μΜ in peripheral retina (1). To assess lower limits of typical for carotenoids, which diminishes to undetectable levels sensitivity, we dissolved lutein and zeaxanthin standards in meth- just a few hundred microns (μm) from the foveal center. Inter- anol at final concentrations of 0, 1, 5, and 10 μΜ and measured estingly, carotenoids are not detectable in photoreceptor outer Raman spectra.