Chemistry Reference
In-Depth Information
For RRI imaging of MP distributions in human subjects we recruited 17 healthy volunteers from
an eye clinic. Laser power levels at the cornea were 4 mW during a measurement; exposure times
were 100 ms for l uorescence measurements, and 300 ms for resonance Raman imaging. The laser
light exposures caused after-images that typically disappeared within a few minutes. During this
time, the setup switched from Raman to l uorescence imaging mode. At a retinal spot size of 3.5 mm
diameter, the photo-thermal light exposure is a factor 16 below the limit set by the ANSI standard
(Sharifzadeh et al. 2008).
When evaluating the MP distributions of all subjects, distinctly different categories are apparent,
as can be seen from representative distributions displayed in Figure 6.8. These feature relatively
wide spatial MP distributions with a high central level, ring-like MP distributions surrounding a
central MP peak, or fragmented distributions. Corresponding intensity line plots along the nasal-
temporal (solid line) and inferior-superior meridians (dotted line), also shown in Figure 6.8, further
highlight the signii cant inter-subject variations in MP levels, symmetries, and spatial extent. The
spatial resolution obtainable with the instrument is approximately sub-50 microns, as can be con-
cluded from the size of small blood vessels discernable in the gray-scale images.
Similar to the case of integrated Raman MP detection, we validated the Raman imaging method
with excised human eyecups. We imaged 11 excised human donor eyecups and compared RRI
derived MP levels with HPLC derived levels (Sharifzadeh et al. 2008). Two-dimensional and three-
dimensional pseudocolor Raman images are shown for two representative eyecups in Figure 6.9a,
with the i rst one featuring a distribution with a relatively strong central peak with a small depres-
sion, and the second one a strongly elongated asymmetrical distribution with high central levels and
relatively smooth decline toward increasing eccentricities. In Figure 6.9e, we plotted the integrated
Raman intensities obtained from the MP RRI images of all eyecups, and compared these optically
derived intensities with HPLC derived MP concentration levels. The result shows a high correlation
between optical and biochemical methods (
R
0.0001).
To further test the RRI imaging method, we compared it with a recently developed, nonmydriatic
version of the lipofuscin l uorescence imaging (autol uorescence imaging) method (Sharifzadeh
et al. 2006). Autol uorescence imaging, AFI, is a less specii c detection method 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 l uorescence and scattering from the optical media
such as the lens (via confocal detection techniques, i ltering, etc.), has to bleach the photoreceptors,
and has to use a location in the peripheral retina as a reference point. The peripheral reference
could potentially lead to an underestimation of the MP density, especially in individuals regularly
consuming high-dose lutein supplements, which can cause substantial increases in even peripheral
carotenoid levels (Bhosale et al. 2007). AFI has an advantage, however, since the peripheral refer-
ence location allows one to eliminate, in i rst order, any potentially confounding attenuation arising
from the anterior optical media.
In Figure 6.10, we summarize the main results of a comparison of MP distributions and concen-
trations obtained with RRI and AFI method for an identical subgroup of subjects. Figure 6.10a and b
compare RRI and AFI obtained for one of the subjects. Compared to the RRI image, the AFI image
is nearly identical, with the exception of a smoother appearance of the distribution. This is due to the
derivation of the MP density map as the logarithm of a ratio between perifoveal and foveal l uores-
cence intensities, which tends to slightly compress the “dynamic range” of the density map ampli-
tudes and smoothen out the resulting MP distribution. For the whole subgroup of 17 subjects, we
integrated the MP levels of images obtained with both methods for each individual over the whole
macula region, and plotted the results in Figure 6.10c. Using a best i t that is not forced through zero,
we obtained a high correlation coefi cient of
R
=
0.92;
p
=
0.89 between both methods. Forcing the i t through
zero, the correlation coefi cient dropped slightly to
R
=
0.80. The high correlation is remarkable in
view of the completely different optical beam paths and derivation methods used to calculate MP
densities in both methods.
=
Search WWH ::
Custom Search