In Vivo Molecular Fluorescence Imaging - Nanotechnology for Biomedical Imaging and Diagnostics - page 268

Therefore, three-dimensional (3D) localization of the fluorophore and quantitative
information about fluorophore concentrations are hard to estimate. For example, two
fluorescence targets located at different depths in a high scattering media project
with different intensity in epi-illumination geometry. on the other hand, the fluores-
cence target can be shadowed or attenuated by an absorbing occlusion on a shallower
depth. Different planar imaging methods are introduced in Section “Modeling of
light propagation in tissue.”
A more accurate method for reconstruction of fluorophore concentrations inside a
medium is fluorescence tomography. Tomography reconstructs the intensity and life-
time of fluorophores inside the tissue by using a light propagation model and
information about positions of the source and detector as well as optical properties of
the medium. More information on tomography methods will be provided in Section
“Imaging algorithms.”

9.4.1 cW domain

The most common fluorescence imaging technique is CW fluorescence imaging.
This method uses a CW or very-low-frequency-modulated light source to provide
the excitation light. The intensity of the reflected and\or transmitted fluorescence
signal is detected by a CCD camera, APD, or PMT. Implementation of this method
is the least expensive and easiest to arrange compared to other fluorescence imaging
techniques. The disadvantage of this method is that it only captures the intensity
information of the fluorescence signals. Captured fluorescence intensity data are
sensitive to the fluctuations of the excitation light, distance of the probe from the
fluorophore, and optical parameters of the system.

9.4.2 td

TD instruments use femtosecond or picosecond laser pulse sources to measure the
temporal distribution of emitted fluorescence light, known as temporal point spread
function (TPSF). The detector should provide a very fast time response and can be
implemented by a time-gated intensified charge-coupled device (ICCD) or a fast
PMT with a time-correlated single-photon counter (TCSPC). TD systems can be used
to reconstruct both fluorescence intensity and lifetime.
Figure 9.10 shows the schematic of a small animal fluorescence imager consisting
of a CW and a TD fluorescence system [42]. A cooled CCD camera with a field of
view of 12 × 12 cm 2 captures the fluorescence images in CW mode with a band-pass
filter (800 ± 20 nm) to find the location of the tumor (the roI) and locate the probe of
the TD system on the roI. The same camera can capture the white-light image of the
small animal after removing the band-pass filter.
The excitation source is a tunable Ti-sapphire pulse laser with a pulse width of
100 fs and repetition rate of 80 MHz (Tsunami, Spectra Physics, Mountain View,
CA). The laser peak can be tuned based on the excitation peak of fluorescent dye.
The femtosecond laser pulse scans the target in a raster pattern through a scanning
head with the one source and four detector fibers at 2-8 mm distances. The integration