Raman Spectroscopy of Ocular Tissue - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 296

giving rise to a broad absorption band ( ∼ 100 nm width) in the blue-green

spectral region, with peak at ∼ 460 nm and clearly resolved vibronic substruc-

1400 cm − 1 , as illustrated in Fig. 12.9b. Following
excitation of the 1 1 B u state, the carotenoid molecule relaxes very rapidly, via
non-radiative transitions, to the lower lying 2 1 A g excited state, from which
electronic emission to the ground state is parity forbidden (Fig. 12.9c). As a
consequence, the luminescence transitions from the 1 1 B u and 2 1 A g states
to the ground states are very weak (10 − 5 -10 − 4 eciency). This allows one to
use resonance Raman spectroscopy (RRS) methods for carotenoid detection.
Compared to non-resonant Raman scattering of carotenoids the RRS cross
section is about five orders of magnitude larger [33]. It becomes possible,
therefore, to explore this method not only for the identification of carotenoids
in their tissue environments but also, through the intensity of the RRS re-
sponse, for the measurement of their tissue concentrations.
The development of Raman-based carotenoid detection for ocular applica-
tions can benefit from already completed previous applications of the method
to human skin [34]. Human skin contains about half a dozen carotenoid species
thought to play a protective role in this largest organ of the human body,
with

tures with a spacing of ∼

-carotene and lycopene having the highest carotenoid concentrations,
and lutein and zeaxanthin playing only a minor role. Since carotenoids are a
good biomarker for fruit and vegetable intake, Raman measurements of skin
carotenoid levels can be used as an indirect rapid, optical method to assess
fruit and vegetable consumption in large populations. These results are of
interest in improving dietary data collected in epidemiological studies, which
in turn are used in developing public health guidelines that promote healthier
diets. The protective effects of diets rich in fruits and vegetables have been
observed for many disease outcomes, including various cancers [35, 36] and
cardiovascular disease [37]. Based on these health benefits, Raman-based skin
carotenoid detection has found large-scale commercial use in the nutritional
supplement industry, where thousands of instruments are in use to monitor
skin uptake of carotenoid-containing multi-vitamin supplements.
Skin carotenoid levels, however, do not correlate with ocular levels of lutein
and zeaxanthin which are likely to be concentrated in the macula by specific
binding proteins. Therefore it is necessary to develop separate detection tech-
nology for the macular pigment measurements.

β

12.7.1 Spatially Integrated Macular Pigment Measurements

In vivo RRS spectroscopy of the macula can take advantage of favorable
anatomical features of the tissue structures encountered in the excitation and
light scattering pathways. The major site of macular carotenoid deposition is
the Henle fiber layer, which has a thickness of only about 100 m, and to a

lesser extent the plexiform layer (Fig. 12.8). Considering that the optical den-
sity of MP in the peak of the absorption band is typically quite a bit smaller
than 1, as determined from direct absorption measurements of MP in excised

μ