Page 230 - Vibrational Spectroscopic Imaging for Biomedical Applications

206 Cha pte r Se v e n

collimating lens, laser line filter, achromatic beam expander, dichroic
beam splitter, and aperture. The laser excitation disk projected onto
the retina has a diameter of ~3.5 mm, and has an intermediate focus
at the position of the aperture. Laser speckle was effectively removed
by mechanically shaking the light delivery fiber. This generates a
spatially homogeneous laser excitation spot via fiber mode mixing. A
red aiming light, coupled into the setup serves as a fixation target for
the subject during a measurement. The optical shutter is designed
such that it allows a small portion of the excitation light to be trans-
mitted even when it is closed, so the subject can view both the red
fixation target and the superimposed excitation disk for optimum
head alignment. The light emitted from the excited disk of the retina
is collimated by the subject’s eye lens and imaged onto a CCD camera
which was kept at a temperature of –10°C during measurements. The
instrument is interfaced to a computer that controls a mechanical
shutter and acquires data for export into appropriate image process-
ing software.
For each measurement two separate images are recorded. In the
first image, the light returned from the retina under 488 nm excitation is
filtered to transmit only 528 nm light, which is the spectral position of
−1
the resonance Raman response of the 1525 cm carbon-carbon double
bond stretch frequency of the MP carotenoids. This is achieved with a
combination of a narrow-bandpass filter (transmission range 528 ± 1
nm), a broader bandpass filter (transmission window 525 ± 15 nm), and
a notch filter. The obtained image contains the Raman response of MP
and overlapping background fluorescence components. In the second
image, “fluorescence image,” the light returned from the retina is fil-
tered with a bandpass filter to only transmit background fluorescence
components slightly above the Raman wavelength, in the 530 to 550 nm
range. As further explained below, this image is subtracted from the
first image to retrieve the pure RRI image of the MP distribution.
For measurements of excised eyecups, the setup is modified as
sketched in inset of Fig. 7.7. In this case, the Raman images are derived
from two measurements taken with a narrow-band tunable filter that
is angle-tuned to “on” and “off” Raman spectral positions.
Human subjects were recruited from an eye clinic and had their eyes
either dilated or undilated depending on their prior eye examination. To
exclude pupil size effects, subjects with dilated eyes were chosen for
intersubject comparisons involving absolute MP levels. Other measure-
ments, such as the identification of a specific type of spatial MP pattern
in a subject’s eye, or the monitoring of the MP distribution in a subject’s
eye over time, were carried out with undilated eyes.
Measurements were carried out in a semi-darkened room. Laser
power levels at the cornea were 4 mW during a measurement; expo-
sure times were 100 ms for background fluorescence measurements,
and 300 ms for resonance Raman imaging. At a retinal spot size of
2
3.5 mm diameter, the area of the exposed retinal field is 0.096 cm , and