Page 245 - Vibrational Spectroscopic Imaging for Biomedical Applications

Raman Detection of Car otenoids in Human T issue 221

since the method only detects the chain’s carbon double-bond vibra-
tion, which is identical in all species. Lycopene has an increased con-
jugation length compared 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
38
independently of the other carotenoid concentrations, and to inves-
tigate specific correlations between lycopene tissue levels and tissue
protection/diseases in future clinical studies.

7.7 Conclusions
In ocular applications, Raman spectroscopy can quickly and objec-
tively assess composite lutein and zeaxanthin concentrations of mac-
ular pigment using spatially averaged, integral, Raman measurements
or Raman images that quantify and map the complete MP distribu-
tion with high-spatial resolution. Importantly, both methods can be
validated with HPLC methods in excised human eyecups and in ani-
mal models.
Both integral and spatially resolved MP Raman methods use the
backscattered, single-path, Raman response from lutein and zeaxan-
thin in the MP containing retinal layer, and largely avoid light tra-
versal through the deeper retinal layers. Since they do not rely on any
reflection of light at the sclera, the overlapping fluorescence signals
from the ocular media can be subtracted from the overall light
response. Importantly, the Raman methods have no assumptions
other than approximating the spectrally broad background fluores-
cence with the fluorescence response at a wavelength that is slightly
offset from the MP Raman response. MP Raman measurements are a
measure of absolute MP concentration levels since the method does
not use a reference point in the peripheral retina. Attenuation effects
caused by the optical media are therefore fully effective, and have to
be avoided or minimized, particularly when comparing MP levels
between subjects. Optical losses from the lens can be neglected in
longitudinal studies provided these are carried out over a time span
in which lens absorptions can be considered to stay constant (1 to
2 years), or in any other studies involving subjects with lens implants.
Therefore, the Raman method would be well suited, for example, in
important nutritional supplementation trials, studies in which sig-
nificant increases in individual MP levels have been demonstrated to
be achievable in a time span of 12 months. 39
Raman imaging clearly reveals the existence of spatially complex
MP distribution patterns throughout the subject population. The dis-
tributions vary strongly regarding widths, axial and rotational
asymmetries, locally depleted areas, and integrated concentration
levels. RRI-derived results provide highly molecule-specific evi-
dence for a more complicated nature of MP distributions in human
subjects than previously thought. It is therefore a valuable method