Page 223 - Vibrational Spectroscopic Imaging for Biomedical Applications

Raman Detection of Car otenoids in Human T issue 199

Here, σ is the Raman scattering cross section, a constant whose
R
magnitude depends only on the excitation and collection geometry.
However, in optically thick media, such as in geometrically thin but
optically dense tissue, a deviation from the linear Raman response of
I versus concentration N can be expected. This could occur, for exam-
s
ple, due to self-absorption of the Stokes Raman signal by the strong
electronic absorption, or due to insufficient excitation light penetra-
tion. In these cases, a nonlinear calibration between RRS response
and molecule concentration may be required using suitable tissue
phantoms.

7.3 Spatially Integrated Resonance Raman
Measurements of Macular Pigment
The human retina can be optically accessed relatively easily. Target-
ing of the macular region with excitation light is simply accomplished
by having the subject fixate on an aiming beam. The excitation and
Raman light have to traverse cornea, aqueous humor, lens, and vitre-
ous, as sketched in Fig. 7.3a. All these ocular media are generally
highly transparent. However, confounding light absorption and scat-
tering can be expected in cases of lens cataracts and in cases of scat-
tering from the vitreous. The macula is essentially free of blood
vessels. When containing a healthy concentration of lutein and zea-
xanthin concentrations, it appears as a gray-shaded area in black-
and-white reflection images of the retina, or in autofluorescence
images taken with blue light, as shown in Fig. 7.3b.
In vivo spectroscopy of the macula can take advantage of favor-
able anatomical features of the tissue structures encountered in the
excitation and Raman light scattering pathways. A cross section of
the retinal tissue layers in the macular region, shown in Fig. 7.4, helps
to illustrate the concept. First, the major sites of macular carotenoid
deposition are the Henle fiber layer, which has a thickness of only
about hundred micrometers, and to a lesser extent the plexiform layer
(both layers are shown in Fig. 7.4 together with the outer nuclear
layer as a single layer, HPN). Considering that the optical density of
MP in the peak of the absorption band is typically lower than 1, as
determined from direct absorption measurements of MP in excised
eyecups, these tissue properties provide essentially an optically thin
film with minimal self-absorption for both the excitation light and the
Raman scattered light, provided the carotenoids/MP are properly
excited on their long-wavelength absorption shoulder. Second, the
Raman scattering approach relies only on the backscattered, single-
path Raman response from the lutein and zeaxanthin-containing MP
layers. Since these retinal layers are located anteriorly to other retinal
layer structures in the optical pathway through the retina, absorption
and fluorescence effects originating from chromophores such as rho-
dopsin in the photoreceptor layer, PhR, and melanin and lipofuscin