Page 232 - Vibrational Spectroscopic Imaging for Biomedical Applications

208 Cha pte r Se v e n

The first term describes fluorescence from the ocular media (OM),
the second term the Raman response from macular pigment (MP),
and the third term fluorescence from lipofuscin (LP). In Eq. (7.1), α
OM
and α are the absorption coefficients of the optical media and lipo-
LP
fuscin, respectively; T and T are the percentage transmissions of
OM MP
the optical media and MP at the indicated absorption and emission
wavelengths; N MP is the concentration of the MP molecules, σ MP
their Raman scattering cross section, and η and η describe the
OM LP
quantum efficiencies for optical media and lipofuscin fluorescence
transitions.
The detector signal at the Raman wavelength is a superposition
of a weak, spectrally narrow Raman signal, and typically 100-fold
stronger and spectrally broad background fluorescence (mostly from
the lens and lipofuscin). To retrieve the MP Raman response, the
superimposed fluorescence intensities need to be measured and sub-
tracted from the total detector signal. The contribution of the broad
fluorescence at the Raman wavelength is approximately the same as
at a wavelength slightly offset to a longer wavelength position, λ .
offset
It can be shown that the Raman component, I (λ ), for each image
R R
pixel is approximately
I (λ ) ≈ T −1 (λ ) T ⋅ −1 (λ I )( (λ )/ T − I (λ / )(T )
R R OM exc OM R Det R R Det o offset offset

where I (λ ) and I (λ ) = detector intensities
Det R Det offset
T and T = filter transmissions at the respective
R offset
wavelengths
T = unknown transmission of the ocular
OM
media
The RRI image of a MP distribution can thus be derived with a
digital image subtraction routine, where the intensities obtained for
each pixel of the two images are divided by the appropriate filter
transmission coefficient.
In order to determine the correct position of the angle tunable
transmission filter, to check for the linearity between resonance
Raman response and optical density of MP, and to determine the
dynamic range of the method, we first measured a number of dried
lutein drops with widely differing concentration levels. All drops
were spotted onto polyvinyl pyrolidine difluoride (PVDF) substrates.
In Fig. 7.9 we show the RRI image obtained for the lutein drop with
the highest concentration level in the center (OD ≈ 0.8). The intensity
of each pixel is color coded according to the linear intensity scale
shown at right. Similar imaging measurements were carried out with
half a dozen drops of varying lutein concentrations. The integrated
response was found to be roughly linear in concentration up to the
highest level.