Biology Reference
In-Depth Information
viability and capacity for high-volume synthesis to support the large-scale
production required for human use.
The primary limitation of fluorescence imaging
in vivo
is the inability to
detect the signal in deep tissues. This limit is imposed by the depth to which
both the excitation light from the illumination source and emission light
from the fluorophore can penetrate the tissue under investigation. Almost
all biological tissues absorb light in the visible to UV range, which limits pen-
etration to only a few hundred micrometers. However, longer excitation
wavelengths (far-red to NIR, i.e., 700-1500 nm) allow the deepest penetra-
tion, that is, up to several centimeters.
47
One of the potential means of circumventing the problems associated
with illumination of tissue is the use of bioluminescent reporters. Biolumi-
nescent proteins such as firefly luciferase (
Photinus pyralis
) occur naturally in a
number of species and emit photos upon catalyzing the oxidation of a sub-
strate and therefore do not rely on an external excitation source. Biolumi-
nescence generally generates a strong signal and high sensitivity in animal
studies; however, because the organism or tissue under study must express
luciferase, these agents are generally restricted to experimental studies in
which a transgenic animal that expresses the enzyme can be generated.
However, examples do exist in which bioluminescent probes have been
used in nontransgenic animals. In this case, the substrate (luciferin) is
administered to the animal (i.p. or i.v.) at a time point after the biolumi-
nescent probe-carrying luciferase has reached the target tissue. After
luciferin administration, the bioluminescent signal is generated within
a short period (1-2 min), peaks at around 10-12 min, and returns to base-
line within an hour.
Regardless of the excitation source, once excited, photons emitted from
the fluorescent molecule have to reach the detector, which is, in most cases,
located outside the body. As is the case of the excitation source, dyes emit-
ting lower energy photons (i.e., NIR 700-900 nm) generally perform better
in terms of absorption by molecules in the body and can be detected at a
depth of several millimeters to a centimeter. A secondary, although ex-
tremely important issue is that of light scattering in the tissue, which reduces
the amount of signal returned and also makes spatial localization of returned
signal problematic. The uncertainty of the signal source becomes greater at
larger tissue depths.
A tertiary issue affecting the quality of information that can be obtained
from
in vivo
fluorescence detection is the autofluorescence of biological tis-
sue. Biomolecules, such as hemoglobin, tryptophan, NADH, pyridoxine,
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