Raman Spectroscopy of Ocular Tissue - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 298

1524 cm − 1 (C=C stretch), 1159 cm − 1
(C-C stretch), and 1007 cm − 1 (C-CH 3 rocking motions) [33]. Spatial resolu-
tion was about 100

characteristic carotenoid Stokes lines at ∼

m. The intensities of the Raman responses were seen to
decrease when probing macular locations of increasing eccentricity, in agree-
ment with the known general spatial concentration profile of the macular
pigments. The linearity of the Raman response with concentration could be
verified by extracting and analyzing tissue carotenoids by high-pressure liquid
chromatography, HPLC, after completion of the Raman measurements [38].
For in vivo experiments and clinical use, several Raman instruments with
lower spectral resolution but highly improved light throughput were developed
[39-41]. A version that is combined with a retinal camera, which permits
independent operator targeting of the subject's macula, is shown in Fig. 12.11a
[42]. The instrument's Raman module, containing a 488 nm laser excitation
source, a spectrograph, and a CCD array detector, is optically connected with
the fundus camera using a beam splitter that is mounted between the front-
end optics of the fundus camera and the eye of the subject. Once alignment is
established, an approximately 1 mm diameter, 1.0 mW, light excitation disk is
projected onto the subject's macula for 0.25 s through the pharmacologically
dilated pupil, and the backscattered light is routed to the Raman module for
detection. Retinal light exposure levels of the instrument are in compliance
with ANSI safety regulations since ocular exposure levels are a factor of 19
below the thermal limit, and a factor of 480 below the photochemical limit
for retinal injury [42].
Typical RRS spectra measured from the macula of a healthy human vol-
unteer through a dilated pupil are displayed, in near real time, on the instru-
ment's computer monitor, as shown in Fig. 12.11b. The left panel shows the
raw spectrum obtained from a single measurement, and clearly reveals the
three characteristic carotenoid Raman signals, which are superimposed on a
steep, spectrally broad fluorescence background. The background is caused
partially by the weak intrinsic fluorescence of lutein and zeaxanthin, and par-
tially by the short-wavelength emission tail of lipofuscin, which is present in
the retinal pigment epithelial layer, and which is excited by the portion of the
excitation light that is transmitted through the MP-containing Henle fiber and
plexiform layers (Fig. 12.8). The ratio between the intensities of the carotenoid
C=C Raman response and the fluorescence background is usually high enough
(

μ

0 . 25) that it is easily possible to quantify the amplitudes of the C=C peak
after digital background subtraction. This step is automatically accomplished
by the instrument's data processing software, which approximates the back-
ground with a fourth-order polynomial, subtracts the background from the
raw spectrum, and displays the final result as processed, scaled, spectrum
in the right panel of the computer monitor, as shown in Fig 12.11b. MP
carotenoid RRS spectra measured for the living human macula were indistin-
guishable from corresponding spectra of pure lutein or zeaxanthin solutions,
measured with the same instrument.
Developed as a portable instrument, the Raman method is currently used
in eye clinics to investigate the role of MP levels in the development of retinal

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