Biology Reference
In-Depth Information
collagen, elastin, flavins, porphyrins, nucleotides, and even water, can absorb
light across the UV-vis range, and some of these molecules emit fluores-
cence that results in attenuation of the fluorophore signal of interest.
Although the natural fluorescence of biological tissue has been put to use
for some clinical applications (as previously described), it is considered a
nuisance variable when attempting to detect the fluorescence signal from
an exogenously administered dye. This problem is reasonably dealt with
by using NIF dyes (790-830 nm emission) because signals emitted from
biological tissue is lower in this wavelength range, or alternatively, by using
special detection methods (time-resolved measurements
48-51
or spectral
unmixing techniques
52,53
.
The inherent intensity of the signal from the fluorophore is also a con-
sideration for
in vivo
imaging, as it directly affects sensitivity and the SNR.
High signal intensity is an obvious benefit for
in vivo
applications because it
reduces the amount of fluorophore that must be administered and also re-
duces the amount of energy deposition needed for dye excitation, thus lim-
iting potential phototoxicity. The signal from a single-fluorophore molecule
is a product of the molecule's quantum yield (QY, i.e., number of photons
emitted per photon absorbed) and its molar extinction coefficient. The sig-
nal will also be affected by optical path length, pH, and the polarity of the
surrounding molecules. In addition, the emitted photons may not contri-
bute to the detected signal as a result of a nonradiative dipole-dipole cou-
pling mechanism such as F
ยจ
rster resonance energy transfer or FRET, with
the surrounding tissue macromolecules, decreasing photon intensity 10-fold
for each centimeter of tissue depth.
54
Some newer classes of engineered pro-
bes such as fluorescent semiconductor nanocrystals (quantum dots, QDs) can
greatly exceed this limit and have demonstrated superior performance for
in
vivo
applications.
55
In addition, QDs are more resistant to photobleaching
and have the added flexibility of being tunable to any given emission fre-
quency. Enthusiasm is somewhat tempered for these agents, however, given
the concerns about their biological safety and their ability to be targeted to
specific tissues.
55
Nonetheless, development of QDs is an active area of re-
search, and expectations are high that QDs with safer toxicity profiles will
become available in time.
Gains in fluorescent signal detection have also been made by advance-
ments in detector technology and computer algorithms for separation and
amplification of the signal. Typically a charge-coupled device (CCD) cam-
era, which is sensitive to a wide range of spectral frequencies, is used to cap-
ture the emitted photons from fluorescence and convert these to a digital
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