Raman Spectroscopy of Ocular Tissue - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 300

Fig. 12.12. Macular pigment levels measured with spatially integrated Raman de-
tection in about 100 volunteer subjects ranging in age between 20 and 50 years.
Each circle represents a subject's MP concentration. MP levels can vary strongly
between subjects

12.7.2 Spatially Resolved Imaging of Macular Pigment

MP distributions are often assumed to have strict rotational symmetry, high
central pigment levels, and a monotonous decline with increasing eccentric-
ity. However, initial resonance Raman imaging, RRI, results obtained with
excised human eyecups demonstrated intriguing deviations, clearly revealing
the existence of strong significant rotational asymmetries, distribution pat-
terns with central depletions, patterns with widely differing widths between
samples, and patterns with fragmented concentration levels [43].
To confirm these “new” distribution features in the living human retina,
the Raman method was developed for in vivo imaging applications [44]. The
experimental setup for this purpose is shown in Fig. 12.13a. Once the subject
achieves head alignment with the help of a red fixation target, as can be seen
in Fig. 12.13b, blue light from a solid state 488 nm laser is projected onto the
macula as a

3 . 5 mm diameter excitation disk, and two images are recorded
with a CCD camera. In the first image, “Raman plus fluorescence image,” the
light returned from the retina under 488 nm excitation is filtered to transmit
only 527 nm light, which is the spectral position of the resonance Raman
response of the 1525 cm − 1 carbon-carbon double bond stretch frequency of
the MP carotenoids. Each pixel of this image contains the Raman response of
MP as well as the fluorescence components overlapping the Raman response at
this wavelength. In the second image, “fluorescence image,” the light returned
from the retina is filtered to only transmit fluorescence components slightly
above the Raman wavelength. The contribution of the broad fluorescence at
the Raman wavelength is approximately the same as at the slightly offset
longer wavelength position, and the RRI image of an MP distribution can
thus be derived with a digital image subtraction routine.

∼