Abstract

We have used resonant Raman scattering as a novel, noninvasive in vivo optical technique to measure the concentration of macular carotenoid pigments in the living human retina. Using a backscattering geometry and resonant molecular excitation in the visible, we measure the Raman peaks that originate from the single- and double-bond stretch vibrations of the π-conjugated molecule’s carbon backbone. The Raman signals scale linearly with carotenoid content, whereas the required laser excitation is well under safety limits for macular exposure. The Raman technique is objective and quantitative and may lead to a new method for rapid screening of carotenoid pigment levels in large human populations that are at risk for vision loss from age-related macular degeneration, the leading cause of blindness of the elderly in the United States.

© 2001 Optical Society of America

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  1. R. A. Bone, J. T. Landrum, and S. L. Tarsis, Vision Res. 25, 1531 (1985).
    [CrossRef]
  2. J. Handelman, D. M. Snodderly, A. J. Adler, M. D. Russett, and E. A. Dratz, Methods Enzymol. 213, 220 (1992).
    [CrossRef]
  3. W. Schalch, P. Dayhaw-Barker, and F. M. Barker, in Nutritional and Environmental Influences on the Eye, A. Taylor, ed. (CRC, Boca Raton, Fla., 1999), p. 215.
  4. D. M. Snodderly, Am. J. Clin. Nutr. Suppl. 62, 1448S (1995).
  5. Eye Disease Case Control Study Group, Arch. Ophthalmol. 111, 104 (1993).
    [CrossRef]
  6. J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
    [CrossRef]
  7. J. T. Landrum, R. A. Bone, and M. D. Kilburn, Adv. Pharmacol. 38, 537 (1997).
    [CrossRef]
  8. B. R. Hammond, B. R. Wooten, and D. M. Snodderly, J. Opt. Soc. Am. A 14, 1187 (1997).
    [CrossRef]
  9. W. Gellermann, M. D. Yoshida, R. W. McClane, N. Balashov, and P. S. Berstein, Proc. SPIE 3250, 8 (1998).
    [CrossRef]
  10. P. S. Bernstein, M. D. Yoshida, N. B. Katz, R. W. McClane, and W. Gellermann, J. Vis. Sci. 39, 2003 (1998).
  11. A. P. Shreve, J. K. Trautman, T. G. Owens, and A. C. Albrecht, Phys. Lett. 178, 89 (1991).
  12. American National Standards Institute, American National Standard for Safe Use of Lasers, (Laser Institute of America, Orlando, Fla., 2000), Sec.  8.3.
  13. Y. Koyama, I. Takatsuka, M. Nakata, and M. Tasumi, J. Raman Spectrosc. 19, 37 (1988).
    [CrossRef]
  14. We achieved the background subtraction by fitting the fluorescence background with a fourth-order polynomial and subtracting it from the composite Raman–fluorescence spectra obtained for each measurement.

1998 (2)

W. Gellermann, M. D. Yoshida, R. W. McClane, N. Balashov, and P. S. Berstein, Proc. SPIE 3250, 8 (1998).
[CrossRef]

P. S. Bernstein, M. D. Yoshida, N. B. Katz, R. W. McClane, and W. Gellermann, J. Vis. Sci. 39, 2003 (1998).

1997 (2)

J. T. Landrum, R. A. Bone, and M. D. Kilburn, Adv. Pharmacol. 38, 537 (1997).
[CrossRef]

B. R. Hammond, B. R. Wooten, and D. M. Snodderly, J. Opt. Soc. Am. A 14, 1187 (1997).
[CrossRef]

1995 (1)

D. M. Snodderly, Am. J. Clin. Nutr. Suppl. 62, 1448S (1995).

1994 (1)

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

1993 (1)

Eye Disease Case Control Study Group, Arch. Ophthalmol. 111, 104 (1993).
[CrossRef]

1992 (1)

J. Handelman, D. M. Snodderly, A. J. Adler, M. D. Russett, and E. A. Dratz, Methods Enzymol. 213, 220 (1992).
[CrossRef]

1991 (1)

A. P. Shreve, J. K. Trautman, T. G. Owens, and A. C. Albrecht, Phys. Lett. 178, 89 (1991).

1988 (1)

Y. Koyama, I. Takatsuka, M. Nakata, and M. Tasumi, J. Raman Spectrosc. 19, 37 (1988).
[CrossRef]

1985 (1)

R. A. Bone, J. T. Landrum, and S. L. Tarsis, Vision Res. 25, 1531 (1985).
[CrossRef]

Adler, A. J.

J. Handelman, D. M. Snodderly, A. J. Adler, M. D. Russett, and E. A. Dratz, Methods Enzymol. 213, 220 (1992).
[CrossRef]

Ajani, U. A.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Albrecht, A. C.

A. P. Shreve, J. K. Trautman, T. G. Owens, and A. C. Albrecht, Phys. Lett. 178, 89 (1991).

Balashov, N.

W. Gellermann, M. D. Yoshida, R. W. McClane, N. Balashov, and P. S. Berstein, Proc. SPIE 3250, 8 (1998).
[CrossRef]

Barker, F. M.

W. Schalch, P. Dayhaw-Barker, and F. M. Barker, in Nutritional and Environmental Influences on the Eye, A. Taylor, ed. (CRC, Boca Raton, Fla., 1999), p. 215.

Bernstein, P. S.

P. S. Bernstein, M. D. Yoshida, N. B. Katz, R. W. McClane, and W. Gellermann, J. Vis. Sci. 39, 2003 (1998).

Berstein, P. S.

W. Gellermann, M. D. Yoshida, R. W. McClane, N. Balashov, and P. S. Berstein, Proc. SPIE 3250, 8 (1998).
[CrossRef]

Blair, N.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Bone, R. A.

J. T. Landrum, R. A. Bone, and M. D. Kilburn, Adv. Pharmacol. 38, 537 (1997).
[CrossRef]

R. A. Bone, J. T. Landrum, and S. L. Tarsis, Vision Res. 25, 1531 (1985).
[CrossRef]

Burton, T. C.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Dayhaw-Barker, P.

W. Schalch, P. Dayhaw-Barker, and F. M. Barker, in Nutritional and Environmental Influences on the Eye, A. Taylor, ed. (CRC, Boca Raton, Fla., 1999), p. 215.

Dratz, E. A.

J. Handelman, D. M. Snodderly, A. J. Adler, M. D. Russett, and E. A. Dratz, Methods Enzymol. 213, 220 (1992).
[CrossRef]

Farber, M. D.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Gellermann, W.

P. S. Bernstein, M. D. Yoshida, N. B. Katz, R. W. McClane, and W. Gellermann, J. Vis. Sci. 39, 2003 (1998).

W. Gellermann, M. D. Yoshida, R. W. McClane, N. Balashov, and P. S. Berstein, Proc. SPIE 3250, 8 (1998).
[CrossRef]

Gragourdas, E. S.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Haller, J.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Hammond, B. R.

Handelman, J.

J. Handelman, D. M. Snodderly, A. J. Adler, M. D. Russett, and E. A. Dratz, Methods Enzymol. 213, 220 (1992).
[CrossRef]

Hiller, R.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Katz, N. B.

P. S. Bernstein, M. D. Yoshida, N. B. Katz, R. W. McClane, and W. Gellermann, J. Vis. Sci. 39, 2003 (1998).

Kilburn, M. D.

J. T. Landrum, R. A. Bone, and M. D. Kilburn, Adv. Pharmacol. 38, 537 (1997).
[CrossRef]

Koyama, Y.

Y. Koyama, I. Takatsuka, M. Nakata, and M. Tasumi, J. Raman Spectrosc. 19, 37 (1988).
[CrossRef]

Landrum, J. T.

J. T. Landrum, R. A. Bone, and M. D. Kilburn, Adv. Pharmacol. 38, 537 (1997).
[CrossRef]

R. A. Bone, J. T. Landrum, and S. L. Tarsis, Vision Res. 25, 1531 (1985).
[CrossRef]

McClane, R. W.

W. Gellermann, M. D. Yoshida, R. W. McClane, N. Balashov, and P. S. Berstein, Proc. SPIE 3250, 8 (1998).
[CrossRef]

P. S. Bernstein, M. D. Yoshida, N. B. Katz, R. W. McClane, and W. Gellermann, J. Vis. Sci. 39, 2003 (1998).

Miller, D. T.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Nakata, M.

Y. Koyama, I. Takatsuka, M. Nakata, and M. Tasumi, J. Raman Spectrosc. 19, 37 (1988).
[CrossRef]

Owens, T. G.

A. P. Shreve, J. K. Trautman, T. G. Owens, and A. C. Albrecht, Phys. Lett. 178, 89 (1991).

Russett, M. D.

J. Handelman, D. M. Snodderly, A. J. Adler, M. D. Russett, and E. A. Dratz, Methods Enzymol. 213, 220 (1992).
[CrossRef]

Schalch, W.

W. Schalch, P. Dayhaw-Barker, and F. M. Barker, in Nutritional and Environmental Influences on the Eye, A. Taylor, ed. (CRC, Boca Raton, Fla., 1999), p. 215.

Seddon, J. M.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Shreve, A. P.

A. P. Shreve, J. K. Trautman, T. G. Owens, and A. C. Albrecht, Phys. Lett. 178, 89 (1991).

Snodderly, D. M.

B. R. Hammond, B. R. Wooten, and D. M. Snodderly, J. Opt. Soc. Am. A 14, 1187 (1997).
[CrossRef]

D. M. Snodderly, Am. J. Clin. Nutr. Suppl. 62, 1448S (1995).

J. Handelman, D. M. Snodderly, A. J. Adler, M. D. Russett, and E. A. Dratz, Methods Enzymol. 213, 220 (1992).
[CrossRef]

Sperduto, R. D.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Takatsuka, I.

Y. Koyama, I. Takatsuka, M. Nakata, and M. Tasumi, J. Raman Spectrosc. 19, 37 (1988).
[CrossRef]

Tarsis, S. L.

R. A. Bone, J. T. Landrum, and S. L. Tarsis, Vision Res. 25, 1531 (1985).
[CrossRef]

Tasumi, M.

Y. Koyama, I. Takatsuka, M. Nakata, and M. Tasumi, J. Raman Spectrosc. 19, 37 (1988).
[CrossRef]

Trautman, J. K.

A. P. Shreve, J. K. Trautman, T. G. Owens, and A. C. Albrecht, Phys. Lett. 178, 89 (1991).

Willet, W.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Wooten, B. R.

Yannuzzi, L. A.

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

Yoshida, M. D.

P. S. Bernstein, M. D. Yoshida, N. B. Katz, R. W. McClane, and W. Gellermann, J. Vis. Sci. 39, 2003 (1998).

W. Gellermann, M. D. Yoshida, R. W. McClane, N. Balashov, and P. S. Berstein, Proc. SPIE 3250, 8 (1998).
[CrossRef]

Adv. Pharmacol. (1)

J. T. Landrum, R. A. Bone, and M. D. Kilburn, Adv. Pharmacol. 38, 537 (1997).
[CrossRef]

Am. J. Clin. Nutr. Suppl. (1)

D. M. Snodderly, Am. J. Clin. Nutr. Suppl. 62, 1448S (1995).

Arch. Ophthalmol. (1)

Eye Disease Case Control Study Group, Arch. Ophthalmol. 111, 104 (1993).
[CrossRef]

J. Am. Med. Assoc. (1)

J. M. Seddon, U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragourdas, J. Haller, D. T. Miller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 242, 1413 (1994).
[CrossRef]

J. Opt. Soc. Am. A (1)

J. Raman Spectrosc. (1)

Y. Koyama, I. Takatsuka, M. Nakata, and M. Tasumi, J. Raman Spectrosc. 19, 37 (1988).
[CrossRef]

J. Vis. Sci. (1)

P. S. Bernstein, M. D. Yoshida, N. B. Katz, R. W. McClane, and W. Gellermann, J. Vis. Sci. 39, 2003 (1998).

Methods Enzymol. (1)

J. Handelman, D. M. Snodderly, A. J. Adler, M. D. Russett, and E. A. Dratz, Methods Enzymol. 213, 220 (1992).
[CrossRef]

Phys. Lett. (1)

A. P. Shreve, J. K. Trautman, T. G. Owens, and A. C. Albrecht, Phys. Lett. 178, 89 (1991).

Proc. SPIE (1)

W. Gellermann, M. D. Yoshida, R. W. McClane, N. Balashov, and P. S. Berstein, Proc. SPIE 3250, 8 (1998).
[CrossRef]

Vision Res. (1)

R. A. Bone, J. T. Landrum, and S. L. Tarsis, Vision Res. 25, 1531 (1985).
[CrossRef]

Other (3)

We achieved the background subtraction by fitting the fluorescence background with a fourth-order polynomial and subtracting it from the composite Raman–fluorescence spectra obtained for each measurement.

American National Standards Institute, American National Standard for Safe Use of Lasers, (Laser Institute of America, Orlando, Fla., 2000), Sec.  8.3.

W. Schalch, P. Dayhaw-Barker, and F. M. Barker, in Nutritional and Environmental Influences on the Eye, A. Taylor, ed. (CRC, Boca Raton, Fla., 1999), p. 215.

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Figures (4)

Fig. 1
Fig. 1

Optical properties of a 7μM solution of lutein in tetrahydrofuran. (a) Absorption spectrum measured through a 1-cm path-length quartz cuvette; inset, energy-level diagram with radiative and nonradiative transitions of the molecule. (b) Luminescence spectrum measured under 488-nm excitation.

Fig. 2
Fig. 2

Experimental setup showing light-delivery and -collection optics of a fiber-based Raman spectrometer interfaced with a fundus camera. M, mirror; other abbreviations defined in text.

Fig. 3
Fig. 3

Raman scattering of lutein tissue spots attached to the retina of a model eye, showing spectra in the carotenoid stretching vibration range for three increasing concentrations: (a) 50, (b) 100, and (c) 200  ng/spot.

Fig. 4
Fig. 4

Raman spectra from the retina of a healthy volunteer, measured with a dilated pupil (8mm diameter). Top, typical spectrum obtained after a single measurement, clearly showing the carotenoid Raman signals superimposed upon a broad fluorescence background. Bottom, Raman spectra summed over three individual measurements before (top trace) and after (bottom trace) subtraction of the fluorescence background.

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