Application of Resonance Raman Spectroscopy to the Detection of Carotenoids In Vivo - Carotenoids: Physical, Chemical, and Biological Functions and Properties - page 101

Laser light

Optical
window

Stratum
corneum

Stratum
granulosum

Epidermis

Stratum
spinosum

Stratum
bosale

Dermis

FIGURE 6.11 Layer structure of human skin as seen in a microscope after staining, showing the morphol-
ogy of dermis, basal layer, stratum spinosum , stratum granulosum , and stratum corneum . Cells of the stra-
tum corneum have no nucleus (these lack the dark staining spots), and form a relatively homogeneous optical
medium, well suited for Raman measurements. For visible wavelengths, the excitation light has a penetration
depth of about 400 μm, and stays within the 0.7-2 mm thick stratum corneum , as indicated.

minimal as well. The penetration depth of visible light into the stratum corneum is approximately
400 microns and therefore is coni ned to this outermost layer, as sketched in Figure 6.11 for a hemi-
spherical beam penetration into the tissue. Using skin tissue sites with thick stratum corneum layer
in RRS measurements, such as the palm of the hand or the sole of the foot, one therefore realizes
measuring conditions of a fairly homogeneous uniform tissue layer with well-dei ned absorption
and scattering conditions.
A i eld-usable instrument coni guration that recently evolved out of the development of RRS for
in vivo skin carotenoid measurements (Gellermann et al. 2001) is shown in Figure 6.12a. It is based
on a miniaturized, i ber-based, and computer-interfaced spectrograph with high light throughput
(Ermakov et al. 2001a). For an RRS skin carotenoid measurement, the palm of the hand is held
against the window of the probe head module and the tissue exposed for about 10 s with 488 nm
laser light at laser intensities of ~10 mW in a 2 mm diameter spot. Carotenoid RRS responses are
detected with a CCD array integrated into the spectrograph. Typical skin carotenoid RRS spectra
measured in vivo are shown in Figure 6.12b. The raw spectrum shown at the top of the panel (trace 1)
was obtained directly after laser exposure and reveals a broad, featureless, strong “autol uores-
cence” background of skin, with three superimposed Raman peaks characteristic for the carotenoid
molecules at 1008, 1159, and 1524 cm −1 . Even though the intensity of the skin l uorescence back-
ground is about 100 times higher than the carotenoid signals, it is possible to measure the skin
carotenoid RRS responses with high accuracy by using a detector with high dynamic range.
Approximation of the l uorescence background with a higher order polynomial and subsequent sub-
traction from the raw spectrum yields an isolated Raman spectrum of the skin carotenoids (trace 2)
that is virtually undistinguishable from a solution of pure -carotene, shown for comparison (trace 3).

The skin carotenoid RRS response originates from contributions of all skin carotenoid species

β