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significant advance since GFP can be used as a reporter of stem cell survival
and differentiation. The challenges of imaging deep structures in the vis-
ible spectrum have been discussed in this chapter. Nevertheless, recent data
suggest that FMT of fluorescent proteins may be possible under certain cir-
cumstances (Garofalakis et al. 2007). Transgenic mice with GFP-expressing T
cells were imaged with a modified FMT system capable of being configured
to produce a very high source-detector density over the target of interest
(Garofalakis et al. 2007). Measurements were performed in reflection mode,
unlike conventional FMT, which uses transillumination to acquire the raw
data. A modified solution of the diffusion equation, accounting for the high
absorption of light in the visible spectrum, was used to derive the Green's
functions for the tomographic reconstruction. With these modifications, GFP-
expressing T cells could be successfully imaged in the thymus and spleen
(Garofalakis et al. 2007). It should be noted, however, that these organs are
fairly superficial and contain large numbers (>10 7 ) of T cells. The ability of
this system to detect realistic numbers of GFP-expressing stem cells in deep
structures, such as the myocardium, remains unlikely. However, significant
effort is being devoted to the generation of red-shifted proteins, which may
be more feasible to image in deep tissues (Deliolanis et al. 2008).
8.4 Imaging Agents
Fluorescent imaging agents can be broadly divided into small organic luo-
rochromes, fluorescent proteins, and quantum dots (QDs) (Frangioni 2003;
Giepmans et al. 2006). Some imaging agents, such as indocyanine green,
can provide a useful readout due to their pharmacokinetic distribution
(Rangaraj et al. 2008; Sevick-Muraca et al. 2008). Other agents produce read-
outs by binding to a specific target (Dumont et al. 2001) or by activation by
a specific enzyme of interest (Mahmood et al. 1999). Targeted fluorescence
agents are frequently substantially larger than PET-detectable agents but
are usually substantially smaller than many MRI-detectable agents, such as
magnetic nanoparticles. The differences in the size and physical properties
of these constructs can have important implications for probe delivery and
pharmacokinetics.
Activatable NIR fluorescent probes have become central to in vivo luo-
rescence imaging and FMT. The NIR fluorochromes on these probes are
held in close physical approximation to each other by peptide linkers, such
as polylysine chains, quenching the fluorescence (Mahmood et al. 1999).
Cleavage of the linker at the recognition site releases the fluorochromes and
produces fluorescence. Activatable agents produce no background signal
until contact with the enzyme of interest and thus constitute an extremely
robust amplification strategy. Increases in signal intensity of up to 100-fold
 
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