Abstract

We describe resonance Raman imaging (RRI) of macular pigment (MP) distributions in the living human eye. MP consists of the antioxidant carotenoid compounds lutein and zeaxanthin, is typically present in high concentrations in the healthy human macula relative to the peripheral retina, and is thought to protect this important central region from age-related macular degeneration. We demonstrate that RRI is capable of quantifying and imaging the spatially strongly varying MP distribution in the human retina. Using laser excitation of the MP molecules at 488nm, and sequential camera detection of light emitted back from the retina at the MP’s strongest Raman peak position and at an off-peak position, RRI maps of MP are obtained at a resolution below 50μm within a fraction of a second per exposure. RRI imaging can be carried out with undilated pupils and provides a highly molecule-specific diagnostic imaging approach for MP distributions in human subjects.

© 2008 Optical Society of America

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  3. Eye Disease Case-Control Study Group, “Antioxidant status and neovascular age-related macular degeneration,” Arch. Ophthalmol. (Chicago) 111, 104-109 (1993). [published correction appears in Arch. Ophthalmol. 111, 1228 (1993), Arch. Ophthalmol. 111, 1366 (1993), and Arch. Ophthalmol. 111, 1499 (1993)].
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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2007 (6)

Age-Related Eye Disease Study Research Group, “The relationship of dietary carotenoid and vitamin A, E, and C intake with age-related macular degeneration in a case-control study,” AREDS Rep. No. 22, Arch. Ophthalmol. (Chicago) 125, 1225-1232 (2007).

M. Trieschmann, S. Beatty, J. M. Nolan, H. W. Hense, B. Heimes, U. Austermann, M. Fobker, and D. Pauleikoff, “Changes in macular pigment optical density and serum concentrations of its constituent carotenoids following supplemental lutein and zeaxanthin: the LUNA study,” Exp. Eye Res. 84, 718-728 (2007).
[CrossRef] [PubMed]

S. Richer, J. Devenport, and J. C. Lang, “LAST II: differential temporal responses of macular pigment optical density in patients with atrophic age-related macular degeneration to dietary supplementation with xanthophylls,” Optometry 78, 213-219 (2007).
[CrossRef] [PubMed]

P. Bhosale, Da You Zhao, and P. S. Bernstein, “HPLC measurement of ocular carotenoid levels in human donor eyes in the lutein supplementation era,” Invest. Ophthalmol. Visual Sci. 48, 543-549 (2007).
[CrossRef]

F. C. Delori, R. H. Webb, and D. H. Sliney, “Maximum permissible exposures for ocular safety (ANSI 2000), with emphasis on ophthalmic devices,” J. Opt. Soc. Am. A 24, 1250-1265 (2007).
[CrossRef]

J. van de Kraatz and D. van Norren, “Optical density of the aging human ocular media in the visible and the UV,” J. Opt. Soc. Am. A 24, 1842-1856 (2007).
[CrossRef]

2006 (4)

T. T. J. M. Berendschot and D. van Norren, “Macular pigment shows ring-like structures,” Invest. Ophthalmol. Visual Sci. 47, 709-714 (2006).
[CrossRef]

S. H. Liew, C. E. Gilbert, T. D. Spector, J. Mellerio, F. J. van Kuijk, S. Beatty, F. Fitzke, J. Marshall, and C. J. Hammond, “Central retinal thickness is positively correlated with macular pigment optical density,” Exp. Eye Res. 82, 915-920 (2006).
[CrossRef]

F. C. Delori, D. G. Goger, C. Keilhauer, P. Salvetti, and G. Staurenghi, “Bimodal spatial distribution of macular pigment: evidence of a gender relationship,” J. Opt. Soc. Am. A 23, 521-538 (2006).
[CrossRef]

M. Sharifzadeh, P. S. Bernstein, and W. Gellermann, “Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions,” J. Opt. Soc. Am. A 23, 2373-2387 (2006).
[CrossRef]

2005 (2)

I. V. Ermakov, M. Sharifzadeh, M. Ermakova, and W. Gellermann, “Resonance Raman detection of carotenoid antioxidants in living human tissue,” J. Biomed. Opt. 10, 064028 (2005).
[CrossRef]

N. I. Krinsky and E. J. Johnson, “Carotenoid actions and their relation to health and disease,” Mol. Aspects Med. 26, 459-516 (2005).
[CrossRef] [PubMed]

2004 (9)

N. Congdon, B. O'Colmain, C. C. Klaver, R. Klein, B. Munoz, D. S. Friedman, J. Kempen, H. R. Taylor, and P. Mitchell, “Causes and prevalence of visual impairment among adults in the United States,” Arch. Ophthalmol. (Chicago) 122, 477-485 (2004).
[CrossRef]

D. S. Friedman, B. J. O'Colmain, B. Munoz, S. C. Tomany, C. McCarty, P. T. de Jong, B. Nemesure, P. Mitchell, and J. Kempen, “Prevalence of age-related macular degeneration in the United States,” Arch. Ophthalmol. (Chicago) 122, 564-572 (2004).

E. Cho, J. M. Seddon, B. Rosner, W. C. Willett, and S. E. Hankinson, “Prospective study of intake of fruits, vegetables, vitamins, and carotenoids and risk for age-related maculopathy,” Arch. Ophthalmol. (Chicago) 122, 883-892 (2004).
[CrossRef]

S. Richer, W. Stiles, L. Statkute, J. Pulido, J. Frankowski, D. Rudy, K. Pei, M. Tsipursky, and J. Nyland, “Double-masked placebo-controlled randomized trial of lutein and antioxidant supplementation in the intervention of atrophic age-related macular degeneration: the Veterans LAST study (Lutein Antioxidant Supplementation Trial),” Optometry 75, 216-230 (2004).
[CrossRef] [PubMed]

R. A. Bone and J. T. Landrum, “Heterochromatic flicker photometry,” Arch. Biochem. Biophys. 430, 137-142 (2004).
[CrossRef] [PubMed]

D. M. Snodderly, J. A. Mares, B. R. Wooten, L. Oxton, M. Gruber, and T. Ficek, “Macular pigment measurement by heterochromatic flicker photometry in older subjects: the carotenoids and age-related eye disease study,” Invest. Ophthalmol. Visual Sci. 45, 531-538 (2004).
[CrossRef]

F. C. Delori, “Autofluorescence method to measure macular pigment optical densities: fluorometry and autofluorescence imaging,” Arch. Biochem. Biophys. 430, 156-162 (2004).
[CrossRef] [PubMed]

I. V. Ermakov, M. R. Ermakova, P. S. Bernstein, and W. Gellermann, “Macular pigment Raman detector for clinical applications,” J. Biomed. Opt. 9, 139-148 (2004).
[CrossRef] [PubMed]

N. P. A. Zagers and D. van Norren, “Absorption of the eye lens and macular pigment derived from reflectance of cone photoreceptors,” J. Opt. Soc. Am. A 21, 2257-2268 (2004).
[CrossRef]

2003 (4)

A. G. Robson, J. D. Moreland, D. Pauleikoff, T. Morrissey, G. E. Holder, F. W. Fitzke, A. D. Bird, and F. J. G. M. D. van Kuijk, “Macular pigment density and distribution: comparison of fundus autofluorescence with minimum motion photometry,” Vision Res. 43, 1765-1775 (2003).
[CrossRef] [PubMed]

M. Trieschmann, G. Spittal, A. Lommartzsch, E. van Kuijk, F. Fitzke, A. C. Bird, and D. Pauleikoff, “Macular pigment: quantitative analysis on autofluorescence images,” Graefe's Arch. Clin. Exp. Ophthalmol. 241, 1006-1012 (2003).
[CrossRef]

C. R. Gale, N. F. Hall, D. I. Phillips, and C. N. Martyn, “Lutein and zeaxanthin status and risk of age-related macular degeneration,” Invest. Ophthalmol. Visual Sci. 44, 2461-2465 (2003).
[CrossRef]

N. I. Krinsky, J. T. Landrum, and R. A. Bone, “Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye,” Annu. Rev. Nutr. 23, 171-201 (2003).
[CrossRef] [PubMed]

2002 (3)

2001 (4)

G. L. Savage, C. A. Johnson, and D. L. Howard, “A comparison of noninvasive objective and subjective measurements of the optical density of human ocular media,” Optom. Vision Sci. 78, 386-395 (2001).
[CrossRef]

F. C. Delori, D. G. Goger, B. R. Hammond, D. M. Snodderly, and S. Burns, “Macular pigment density measured by autofluorescence spectrometry: comparison with reflectometry and heterochromatic flicker photometry,” J. Opt. Soc. Am. A 18, 1212-1230 (2001).
[CrossRef]

J. T. Landrum and R. A. Bone, “Lutein, zeaxanthin, and the macular pigment,” Arch. Biochem. Biophys. 385, 28-40 (2001).
[CrossRef] [PubMed]

S. Beatty, I. J. Murray, D. B. Henson, D. Carden, H. Koh, and M. E. Boulton, “Macular pigment and risk for age-related macular degeneration in subjects from a Northern European population,” Invest. Ophthalmol. Visual Sci. 42, 439-446 (2001).

2000 (1)

1998 (1)

A. E. Elsner, S. A. Burns, E. Beausencourt, and J. J. Weiter, “Foveal cone photopigment distribution: small alterations associated with macular pigment distribution,” Invest. Ophthalmol. Visual Sci. 39, 2394-2404 (1998).

1997 (1)

1994 (1)

Eye Disease Case-Control Study Group, “Dietary carotenoids, vitamins A, C, and E, and advanced age-related macular degeneration,” J. Am. Med. Assoc. 272, 1413-1420 (1994).
[CrossRef]

1993 (1)

Eye Disease Case-Control Study Group, “Antioxidant status and neovascular age-related macular degeneration,” Arch. Ophthalmol. (Chicago) 111, 104-109 (1993). [published correction appears in Arch. Ophthalmol. 111, 1228 (1993), Arch. Ophthalmol. 111, 1366 (1993), and Arch. Ophthalmol. 111, 1499 (1993)].

1986 (1)

1984 (2)

D. M. Snodderly, P. K. Brown, F. C. Delori, and J. D. Auran, “The macular pigment. I. Absorbance spectra, localization, and discrimination from other yellow pigments in primate retinas,” Invest. Ophthalmol. Visual Sci. 25, 660-673 (1984).

D. M. Snodderly, J. D. Auran, and F. C. Delori, “The macular pigment. II. Spatial distribution in primate retinas,” Invest. Ophthalmol. Visual Sci. 25, 674-685 (1984).

Annu. Rev. Nutr. (1)

N. I. Krinsky, J. T. Landrum, and R. A. Bone, “Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye,” Annu. Rev. Nutr. 23, 171-201 (2003).
[CrossRef] [PubMed]

Appl. Opt. (1)

Arch. Biochem. Biophys. (3)

F. C. Delori, “Autofluorescence method to measure macular pigment optical densities: fluorometry and autofluorescence imaging,” Arch. Biochem. Biophys. 430, 156-162 (2004).
[CrossRef] [PubMed]

R. A. Bone and J. T. Landrum, “Heterochromatic flicker photometry,” Arch. Biochem. Biophys. 430, 137-142 (2004).
[CrossRef] [PubMed]

J. T. Landrum and R. A. Bone, “Lutein, zeaxanthin, and the macular pigment,” Arch. Biochem. Biophys. 385, 28-40 (2001).
[CrossRef] [PubMed]

Arch. Ophthalmol. (Chicago) (5)

Eye Disease Case-Control Study Group, “Antioxidant status and neovascular age-related macular degeneration,” Arch. Ophthalmol. (Chicago) 111, 104-109 (1993). [published correction appears in Arch. Ophthalmol. 111, 1228 (1993), Arch. Ophthalmol. 111, 1366 (1993), and Arch. Ophthalmol. 111, 1499 (1993)].

Age-Related Eye Disease Study Research Group, “The relationship of dietary carotenoid and vitamin A, E, and C intake with age-related macular degeneration in a case-control study,” AREDS Rep. No. 22, Arch. Ophthalmol. (Chicago) 125, 1225-1232 (2007).

N. Congdon, B. O'Colmain, C. C. Klaver, R. Klein, B. Munoz, D. S. Friedman, J. Kempen, H. R. Taylor, and P. Mitchell, “Causes and prevalence of visual impairment among adults in the United States,” Arch. Ophthalmol. (Chicago) 122, 477-485 (2004).
[CrossRef]

D. S. Friedman, B. J. O'Colmain, B. Munoz, S. C. Tomany, C. McCarty, P. T. de Jong, B. Nemesure, P. Mitchell, and J. Kempen, “Prevalence of age-related macular degeneration in the United States,” Arch. Ophthalmol. (Chicago) 122, 564-572 (2004).

E. Cho, J. M. Seddon, B. Rosner, W. C. Willett, and S. E. Hankinson, “Prospective study of intake of fruits, vegetables, vitamins, and carotenoids and risk for age-related maculopathy,” Arch. Ophthalmol. (Chicago) 122, 883-892 (2004).
[CrossRef]

Exp. Eye Res. (2)

M. Trieschmann, S. Beatty, J. M. Nolan, H. W. Hense, B. Heimes, U. Austermann, M. Fobker, and D. Pauleikoff, “Changes in macular pigment optical density and serum concentrations of its constituent carotenoids following supplemental lutein and zeaxanthin: the LUNA study,” Exp. Eye Res. 84, 718-728 (2007).
[CrossRef] [PubMed]

S. H. Liew, C. E. Gilbert, T. D. Spector, J. Mellerio, F. J. van Kuijk, S. Beatty, F. Fitzke, J. Marshall, and C. J. Hammond, “Central retinal thickness is positively correlated with macular pigment optical density,” Exp. Eye Res. 82, 915-920 (2006).
[CrossRef]

Graefe's Arch. Clin. Exp. Ophthalmol. (1)

M. Trieschmann, G. Spittal, A. Lommartzsch, E. van Kuijk, F. Fitzke, A. C. Bird, and D. Pauleikoff, “Macular pigment: quantitative analysis on autofluorescence images,” Graefe's Arch. Clin. Exp. Ophthalmol. 241, 1006-1012 (2003).
[CrossRef]

Invest. Ophthalmol. Visual Sci. (8)

T. T. J. M. Berendschot and D. van Norren, “Macular pigment shows ring-like structures,” Invest. Ophthalmol. Visual Sci. 47, 709-714 (2006).
[CrossRef]

P. Bhosale, Da You Zhao, and P. S. Bernstein, “HPLC measurement of ocular carotenoid levels in human donor eyes in the lutein supplementation era,” Invest. Ophthalmol. Visual Sci. 48, 543-549 (2007).
[CrossRef]

D. M. Snodderly, J. A. Mares, B. R. Wooten, L. Oxton, M. Gruber, and T. Ficek, “Macular pigment measurement by heterochromatic flicker photometry in older subjects: the carotenoids and age-related eye disease study,” Invest. Ophthalmol. Visual Sci. 45, 531-538 (2004).
[CrossRef]

A. E. Elsner, S. A. Burns, E. Beausencourt, and J. J. Weiter, “Foveal cone photopigment distribution: small alterations associated with macular pigment distribution,” Invest. Ophthalmol. Visual Sci. 39, 2394-2404 (1998).

D. M. Snodderly, P. K. Brown, F. C. Delori, and J. D. Auran, “The macular pigment. I. Absorbance spectra, localization, and discrimination from other yellow pigments in primate retinas,” Invest. Ophthalmol. Visual Sci. 25, 660-673 (1984).

D. M. Snodderly, J. D. Auran, and F. C. Delori, “The macular pigment. II. Spatial distribution in primate retinas,” Invest. Ophthalmol. Visual Sci. 25, 674-685 (1984).

C. R. Gale, N. F. Hall, D. I. Phillips, and C. N. Martyn, “Lutein and zeaxanthin status and risk of age-related macular degeneration,” Invest. Ophthalmol. Visual Sci. 44, 2461-2465 (2003).
[CrossRef]

S. Beatty, I. J. Murray, D. B. Henson, D. Carden, H. Koh, and M. E. Boulton, “Macular pigment and risk for age-related macular degeneration in subjects from a Northern European population,” Invest. Ophthalmol. Visual Sci. 42, 439-446 (2001).

J. Am. Med. Assoc. (1)

Eye Disease Case-Control Study Group, “Dietary carotenoids, vitamins A, C, and E, and advanced age-related macular degeneration,” J. Am. Med. Assoc. 272, 1413-1420 (1994).
[CrossRef]

J. Biomed. Opt. (2)

I. V. Ermakov, M. Sharifzadeh, M. Ermakova, and W. Gellermann, “Resonance Raman detection of carotenoid antioxidants in living human tissue,” J. Biomed. Opt. 10, 064028 (2005).
[CrossRef]

I. V. Ermakov, M. R. Ermakova, P. S. Bernstein, and W. Gellermann, “Macular pigment Raman detector for clinical applications,” J. Biomed. Opt. 9, 139-148 (2004).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (8)

W. Gellermann, I. V. Ermakov, M. R. Ermakova, R. W. McClane, D.-Y. Zhao, and P. S. Bernstein, “In vivo resonant Raman measurement of macular carotenoid pigments in the young and the aging human retina,” J. Opt. Soc. Am. A 19, 1172-1186 (2002).
[CrossRef]

N. P. A. Zagers and D. van Norren, “Absorption of the eye lens and macular pigment derived from reflectance of cone photoreceptors,” J. Opt. Soc. Am. A 21, 2257-2268 (2004).
[CrossRef]

F. C. Delori, D. G. Goger, C. Keilhauer, P. Salvetti, and G. Staurenghi, “Bimodal spatial distribution of macular pigment: evidence of a gender relationship,” J. Opt. Soc. Am. A 23, 521-538 (2006).
[CrossRef]

M. Sharifzadeh, P. S. Bernstein, and W. Gellermann, “Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions,” J. Opt. Soc. Am. A 23, 2373-2387 (2006).
[CrossRef]

F. C. Delori, R. H. Webb, and D. H. Sliney, “Maximum permissible exposures for ocular safety (ANSI 2000), with emphasis on ophthalmic devices,” J. Opt. Soc. Am. A 24, 1250-1265 (2007).
[CrossRef]

J. van de Kraatz and D. van Norren, “Optical density of the aging human ocular media in the visible and the UV,” J. Opt. Soc. Am. A 24, 1842-1856 (2007).
[CrossRef]

F. C. Delori, D. G. Goger, B. R. Hammond, D. M. Snodderly, and S. Burns, “Macular pigment density measured by autofluorescence spectrometry: comparison with reflectometry and heterochromatic flicker photometry,” J. Opt. Soc. Am. A 18, 1212-1230 (2001).
[CrossRef]

B. R. Hammond, B. R. Wooten, and D. M. Snodderly, “Individual variations in the spatial profile of human macular pigment,” J. Opt. Soc. Am. A 14, 1187-1196 (1997).
[CrossRef]

Med. Phys. (1)

Y. Chang, F. L. Lee, S. J. Chen, and S. F. Chen, “Optical measurement of human retinal macular pigment and its spatial distribution with age,” Med. Phys. 29, 2621-2628 (2002).
[CrossRef] [PubMed]

Mol. Aspects Med. (1)

N. I. Krinsky and E. J. Johnson, “Carotenoid actions and their relation to health and disease,” Mol. Aspects Med. 26, 459-516 (2005).
[CrossRef] [PubMed]

Opt. Express (1)

Opt. Lett. (1)

Optom. Vision Sci. (1)

G. L. Savage, C. A. Johnson, and D. L. Howard, “A comparison of noninvasive objective and subjective measurements of the optical density of human ocular media,” Optom. Vision Sci. 78, 386-395 (2001).
[CrossRef]

Optometry (2)

S. Richer, W. Stiles, L. Statkute, J. Pulido, J. Frankowski, D. Rudy, K. Pei, M. Tsipursky, and J. Nyland, “Double-masked placebo-controlled randomized trial of lutein and antioxidant supplementation in the intervention of atrophic age-related macular degeneration: the Veterans LAST study (Lutein Antioxidant Supplementation Trial),” Optometry 75, 216-230 (2004).
[CrossRef] [PubMed]

S. Richer, J. Devenport, and J. C. Lang, “LAST II: differential temporal responses of macular pigment optical density in patients with atrophic age-related macular degeneration to dietary supplementation with xanthophylls,” Optometry 78, 213-219 (2007).
[CrossRef] [PubMed]

Vision Res. (1)

A. G. Robson, J. D. Moreland, D. Pauleikoff, T. Morrissey, G. E. Holder, F. W. Fitzke, A. D. Bird, and F. J. G. M. D. van Kuijk, “Macular pigment density and distribution: comparison of fundus autofluorescence with minimum motion photometry,” Vision Res. 43, 1765-1775 (2003).
[CrossRef] [PubMed]

Other (3)

P. S. Bernstein, Moran Eye Center, University of Utah, 65 Medical Drive, Salt Lake City, Utah 84132 (personal communication, 2007).

American National Standards Institute, “American national standard for safe use of lasers,” ANSI Z136.1-2000 (Laser Institute of America, 2000), Sec. 8.3.

The angular field diameter subtended by the excitation spot is 206 mrad, and for the CE parameter in Table 6, ANSI standard one obtains CE=α2/(αmaxαmin)=(206)2/(100×1.5)=283. According to paragraph 8.3 of ANSI, since t=0.4s, one can use an exposure time t=0.07. For the maximum permissible radiant exposure at the cornea, one then obtains CE×1.8×t0.75×10−3=69.3mJ/cm2, and for the maximum permissible radiant exposure at the retina 69.3mJ/cm2×(pupil area/retinal area)=69.3mJ/cm2×(7/3.5)2=277mJ/cm2.

Supplementary Material (1)

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Figures (9)

Fig. 1
Fig. 1

Schematics of RRI system used to measure MP distributions in the living human eye and in excised retinal sample preparations. A 488 nm excitation beam from a solid-state laser is routed onto the retina via an optical fiber, collimating lens, L1, laser line filter, F1, achromatic beam expander, L2, dichroic beam splitter, BS1, and aperture, AP. Beam spot size on the retina is 3.5 mm in diameter. Light emitted from the retina is imaged onto the pixel array of a CCD camera with a 50 mm achromat. For RRI imaging, the light returned from the retina is first filtered with a combination of a narrowband tunable filter, F4, a bandpass filter, F5, and a notch filter, F3, effectively limiting the transmitted wavelength range to 528 nm , the spectral position of the resonance Raman response of MP under 488 nm excitation. In order to determine a correction for the background fluorescence, the light returned from the retina is filtered, in a second image, with a bandpass filter centered in the vicinity of the Raman peak at 540 nm . Inset (a) shows the modification of the setup for measurements of excised eyecups, which are Raman imaged with the help of an angle-tunable filter, F4, tuned to two “on” and “off” Raman peak spectral positions near 528 nm . Inset (b) shows the modification for RRI imaging of retinal tissue sections through a microscope. Samples can be viewed with white-light illumination and translated with micrometer-scale precision.

Fig. 2
Fig. 2

Schematics of retinal layer system with indication of excitation and emission light intensities encountered in RRI of MP: OM, anterior optical media; MP, macular-pigment-containing layer; OS, photoreceptor outer segment layer; LP, lipofuscin-containing layer; I O M , light intensity originating from optical media fluorescence; I R , Raman response from MP; I L P , light intensity due to lipofuscin fluorescence (see text).

Fig. 3
Fig. 3

En face (a) and three-dimensional (b) pseudocolor-scaled RRI images of a dried drop of lutein spotted onto a PVDF substrate. The lutein solution had a physiological carotenoid concentration OD 0.8 . The intensity of each pixel is color coded according to the scale shown in (a) and is displayed as a function of pixel position in the camera CCD array (1 pixel= 20 μ m ).

Fig. 4
Fig. 4

(a), (c) Two-dimensional, pseudocolor Raman images of 2 of the 11 donor eyecups imaged to establish a correlation between Raman- and HPLC-derived carotenoid levels; (b), (d) corresponding three-dimensional images. The color scale bar indicates the color coding of light intensities. The graph in (e) shows the correlation between optical intensities integrated over the macular regions of the eyecups and subsequently derived HPLC levels obtained for 8 mm diameter tissue punches centered on the macula (correlation coefficient R = 0.92 ; p < 0.0001 ).

Fig. 5
Fig. 5

RRI results for MP distribution in living human eye. (a) Typical gray-scale image obtained after subtraction of fluorescence background from pixel intensity map containing Raman response and superimposed fluorescence background. (b) Pseudocolor-scaled, three-dimensional representation of gray-scale image. (c) Line plots along nasal–temporal (solid curve) and inferior–superior meridians (dashed curve), both running through the center of the MP distribution.

Fig. 6
Fig. 6

(a) Pseudocolor-scaled, three-dimensional MP RRI images obtained from four healthy subjects, demonstrating significant intersubject variations in MP levels, symmetries, and spatial extent. Note ringlike MP distribution with small central MP peak and overall low levels in one of the cases (lower left). All images are color coded with the same intensity bar. (b) Intensity plot profiles derived for each MP distribution from pixel intensity maps ( 22 × 1400 μ m rectangles) running along nasal–temporal meridians through the center of the macula.

Fig. 7
Fig. 7

Images of MP distributions obtained for the same subject with (a) RRI and (b) fluorescence-based imaging. (c) Comparison of integrated MP densities obtained for 17 subjects with Raman- and fluorescence-based imaging methods. Vertical scale shows integrated MP densities derived from RRI images by integrating intensities over the whole macular region; horizontal scale shows corresponding densities derived via fluorescence imaging. A high correlation coefficient of R = 0.89 is obtained for both methods, using a best fit that is not forced through zero. If the fit is forced through zero (not shown), one obtains a correlation coefficient R = 0.80 .

Fig. 8
Fig. 8

Optical density in fovea versus thickness of retina in foveal pit, measured for eight healthy subjects. Thickness data were obtained from optical coherence tomography scans (see text).

Fig. 9
Fig. 9

Pseudocolor RRI images of three subjects with ringlike MP distributions. (a) (Multimedia online;josaa.osa.org) A 57-year-old healthy male with MP distribution consisting of a narrow central peak and a surrounding strong, nearly rotationally symmetric distribution. MP levels in the ring are slightly higher than in the center and feature a noticeable disruption/offset at the “2 o’clock” position. (b) A 70-year-old female diagnosed with a mild form of dry AMD, showing a weak, broken-up ring structure with central high MP density and crosslike spokes. (c), (d) MP distributions in left (c) and right eye (d) of a 62-year-old female measured after detachment of the vitreous in the right eye. Six months prior to detachment, RRI images revealed the same ringlike MP pattern with a central spike in both eyes. Detachment of the vitreous apparently caused the formation of a double-peak MP structure inside the MP ring in this subject.

Tables (1)

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Table 1 Demographics of the Population

Equations (5)

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I D e t ( λ R ) = α O M ( λ e x c ) η O M ( λ R ) I ( λ e x c ) + T O M ( λ e x c ) T O M ( λ R ) N M P σ M P ( λ R ) I ( λ e x c ) + T O M ( λ e x c ) T M P ( λ e x c ) α L P ( λ e x c ) η L P ( λ R ) I ( λ e x c ) T M P ( λ R ) T O M ( λ R ) .
I D e t ( λ o f f s e t ) α O M ( λ e x c ) η O M ( λ R ) I ( λ e x c ) + T O M ( λ e x c ) T M P ( λ e x c ) α L P ( λ e x c ) η L P ( λ R ) I ( λ e x c ) T M P ( λ R ) T O M ( λ R ) .
I R ( λ R ) N M P σ M P ( λ R ) I ( λ e x c ) ,
I R ( λ R ) T O M 1 ( λ e x c ) T O M 1 ( λ R ) ( I D e t ( λ R ) I D e t ( λ o f f s e t ) ) .
I R ( λ R ) T O M 1 ( λ e x c ) T O M 1 ( λ R ) ( I D e t ( λ R ) T R I D e t ( λ o f f s e t ) T o f f s e t ) .

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