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Raman Detection of Car otenoids in Human T issue 211

plot the spatially integrated Raman intensities obtained from the MP RRI
images of all eyecups, and compare these optically derived intensities
with HPLC-derived MP concentration levels. A high correlation is
obtained between optical and biochemical method (R = 0.92; p = 0.0001).
To further test the RRI imaging method, we compared it with a
recently developed, nonmydriatic version of the lipofuscin fluores-
27
cence imaging (“autofluorescence imaging”) method. Autofluores-
cence imaging, AFI, is a less specific detection methods since it detects
the light emitted from a compound other than MP, and thus derives
the concentration of MP only indirectly. The method has to take into
account light traversal through deeper retinal layers, has to carefully
eliminate image contrast diminishing fluorescence and scattering
from the optical media like the lens (via confocal detection techniques,
filtering, etc.), has to bleach the photoreceptors, and has to use a loca-
tion in the peripheral retina as a reference point. The peripheral refer-
ence could potentially lead to an underestimation of the MP density,
especially in individuals regularly consuming high-dose lutein sup-
plements, which can cause substantial increases in even peripheral
29
carotenoid levels. AFI has an advantage, however, since the periph-
eral reference location allows one to eliminate, in first order, any poten-
tially confounding attenuation arising from anterior optical media, and
the instrumentation is less complex compared to RRI imaging.

7.5 Spatially Resolved Resonance Raman Imaging of
Macular Pigment in Human Subjects
For RRI imaging of MP distributions in human subjects we recruited
17 healthy volunteers. All procedures were performed with Institu-
tional Review Board (IRB) approval and were in accordance with the
tenets of the Declaration of Helsinki. Imaging details for one of the
subjects are shown in Fig. 7.11. Figure 7.11a shows the gray-scale image
obtained after subtraction of the fluorescence background, Fig. 7.11b a
pseudo-color, three-dimensional representation of the distribution,
and Fig. 7.11c two-line plots along the nasal-temporal and inferior-
superior meridians, respectively. The MP distribution in this subject
features a wide, axially slightly asymmetric distribution with rela-
tively high-central MP levels, and a monotonous decline of MP levels
toward the peripheral retina. The spatial resolution obtainable with the
instrument is approximately sub-50-μm as can be determined from
the size of small blood vessels discernable in the gray-scale images.
To check whether the obtained imaging results depend on the
degree of bleaching of the photoreceptors, we compared two images
of a subject’s retina, one obtained after exposing it to blue bleaching
light for 2 minutes, and the other obtained after no bleaching. The
resulting images were seen to be identical. Evaluating the MP distri-
butions of all subjects, distinctly different categories are clearly
revealed. Representative distributions are shown in Fig. 7.12a. One is