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to the other carotenoids in skin and therefore features a small (~10 nm) but distinguishable red
shift of the absorption. This shift can be explored to measure skin lycopene levels independently
of the other carotenoid concentrations (Ermakov et al. 2004b). For pure solutions of lycopene and
β
-carotene, the resonance Raman response has approximately the same strengths under 488 nm
excitation. Under excitation with 514.5 nm, however, the response is about six times higher for
lycopene. Taking this effect into account in a simple two-carotenoid model, it is possible to derive
skin lycopene concentrations separately by measuring two RRS responses, one for 488 nm excita-
tion, and one for 514.5 nm excitation (Ermakov et al. 2004b). For the ratio of the two concentrations,
N
B
/
N
L
, where
N
B
is the concentration of all carotenoids other than lycopene and
N
L
is the lycopene
concentration, one obtains Equation 6.3
N
r
σ−σ
488
r
514
B
L
L
=
(6.3)
514
488
N
σ−σ
L
B
B
where
r
I
488
/
I
514
is the ratio of the RRS responses for blue and green excitation, respectively
σ
j
i
is the respective Raman cross sections of the two carotenoid species
=
The RRS instrument for the selective detection of dermal lycopene levels is shown in Figure 6.15.
The instrument uses a single spectrograph to detect C
C Raman responses resulting from 488 and
514 nm excitation with a i xed grating position. A small air-cooled, multiline argon laser generates
excitation light at both wavelengths with comparable intensities. Two shutters are synchronized
such that the skin is either unexposed, exposed with 488 nm light, or with 514 nm light. The optical
probe module contains an additional “green” excitation channel, and the detection channels each
contain a separate i lter to suppress scattered excitation light. A measurement starts by exposing
the skin site with 488 nm, while recording the RRS carotenoid C
=
C response. Subsequently, the
electronics closes the shutter, reads out the Raman data, reactivates the CCD, and the whole process
is repeated for 514 nm green excitation. Finally, the software calculates and separately displays the
ratio of the carotenoids and the skin lycopene levels, as shown in Figure 6.15b.
For seven volunteer subjects measured with the dual-wavelength RRS instrument, we obtained
the skin carotenoid RRS results shown in Figure 6.16, where the individual lycopene and carotene
levels are indicated together with the lycopene/carotene ratio for each subject. Interestingly, there
is a strong, almost threefold variation in carotene to lycopene ratio in the measured subjects, rang-
ing from 0.54 to 1.55. This means that substantially different carotenoid compositions can exist in
human skin, with some subjects exhibiting almost twice the concentration of lycopene compared
to carotene, and other subjects showing the opposite effect. This behavior could rel ect different
dietary patterns regarding the intake of lycopene or lycopene-containing vegetables, or it could
point toward differing abilities between subjects to accumulate these carotenoids in the skin.
=
6.7 CONCLUSIONS
In ocular applications, Raman spectroscopy can quickly and objectively assess composite lutein
and zeaxanthin concentrations of macular pigment using spatially averaged, integral measure-
ments or images that quantify and map the complete MP distribution with high spatial resolution.
Importantly, both variants can be validated with HPLC methods in excised human eyecups and in
animal models.
Both integral and spatially resolved MP Raman methods use the backscattered, single-path
Raman response from lutein and zeaxanthin in the MP-containing retinal layer, and largely avoid
light traversal through the deeper retinal layers. Since they do not rely on any rel ection of light at the
sclera, the overlapping l uorescence signals from the ocular media can be subtracted from the over-
all light response. Importantly, the Raman methods make no assumptions other than approximating
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