Page 218 - Vibrational Spectroscopic Imaging for Biomedical Applications

194 Cha pte r Se v e n

in living human tissue. Raman spectroscopy is a well-known, highly
molecule specific form of vibrational spectroscopy that is commonly
used to identify molecular compounds through their spectrally narrow
“Raman fingerprint” responses. Most frequently, off-resonance Raman
techniques are used for this purpose since they avoid the strong intrin-
sic electronic fluorescence transitions typically encountered in complex
molecules. Carotenoid molecules, however, possess a unique energy
level structure and associated optical pumping cycle. While easily
excited from the ground state into a higher excited state within a strong,
electric dipole-allowed absorption transition, they relax quickly into a
new, lower-lying excited state, from which fluorescence transitions back
to the ground state are forbidden. This offers the opportunity to use
fluorescence-background-free resonant excitation of the carotenoids in
their visible absorption bands, which results in a resonance enhance-
ment of the carotenoid Raman response by about five orders of magni-
1
tude relative to nonresonant Raman scattering. It becomes possible,
therefore, to explore RRS not only for the identification of carotenoids in
biological tissue environments, but also, through the intensity of their
RRS response, for the quantification of their tissue concentrations. The
tissue environment can be expected to have only a minor effect on the
molecule’s vibrational energy. This should cause the Raman signature
to be virtually identical for the isolated carotenoid molecule, the mole-
cule in solution, or the molecule in a cell environment. However, the
applicability of the method can be expected to depend heavily on poten-
tially confounding tissue properties, such as a saturation of the carot-
enoid Raman response at high concentrations, or the existence of other
molecules with potentially interfering scattering, absorption and/or
fluorescence contributions. A crucial task therefore is the validation of
the RRS detection method for the particular tissue environment. If suc-
cessful, RRS could be used as a novel optical diagnostic method for the
measurement of tissue carotenoid levels, potentially allowing one to
measure large populations in clinical and field settings, and to track
their changes occurring over time as a consequence of developing
pathology and/or tissue uptake.
A tissue site that is particularly interesting for the application of
the Raman method is the macula lutea. It is the well-known “yellow
spot” in the human retina that contains the highest concentration of
carotenoids in the human body. Of the approximately 10 carotenoid
species identified in human serum, only two carotenoids, lutein and
zeaxanthin, are selectively taken up at this retinal tissue site. Their
concentrations can be as high as several 10 ng/g of tissue, however, in
the healthy human retina. Due to their strong absorption in the blue-
green spectral range, the macular carotenoids, also termed macular
pigment (MP) impart a yellow coloration to the macula. When viewing
the retinal in cross section, MP is located anterior to the photoreceptor
2,3
outer segments and the retinal pigment epithelium. It is therefore
thought to shield these vulnerable tissues from light-induced oxidative