Rational Design of Fluorophores for In Vivo Applications - Progress in Molecular Biology and Translational Science - page 92

hydrocarbons or small fluorescent heterocycles) in a DNA-like
phosphodiester oligomer (oligodeoxyfluorosides). They found that several
different interactions between the assembled fluorophores occur (such as
excimer and exciplex formation, H-stacking and energy transfer), which
result in different fluorescence characteristics for different combinations of
the fluorophores. This enabled preparing the sets of short oligomers
(containing 1-4 different fluorophores) each with a different fluorescence
color, excitable at the common wavelength. The advantages of this
approach are the ease of synthesis (as an automated DNA synthesizer can
be utilized), water solubility (provided by phosphodiester backbone), and
the ability to attach a single bioconjugatable group. 241 The
antibody-oligodeoxyfluoroside conjugates have been used for multicolor
imaging of living cells 241 and living zebrafish embryo. 240
6.2. Self-illuminating fluorophores
All the fluorophores described so far require photoexcitation prior to fluo-
rescence, that is, the fluorophore must absorb a photon to be excited, which
in turn requires illumination of the fluorophore by an external source of
light. Such illumination causes excitation of the exogenous tissue or cell
fluorophores and causes some background signal which reduces the
signal-to-background ratio. Consequently, it diminishes both the sensitivity
and the limit of detection, even when the excitation is done in the optimal
spectral window. 242
One of the solutions to overcome this problem is to use fluorophores that
do not require photoexcitation but can reach an excited state and subse-
quently emit fluorescence upon chemical reaction (chemiluminescence)
or upon enzymatic reaction (bioluminescence). Chemi- or bioluminescence
eliminates the need for an external light source for excitation and therefore
eliminates almost completely autofluorescence from the tissue.
The prominent bioluminescent reaction is the luciferase-catalyzed oxi-
dation of luciferin with the concomitant emission of light. Luciferin is a ge-
neric name given to the class of small organic molecules that can emit light
upon oxidation catalyzed by various types of luciferases, among which firefly
luciferin and coelenterazine are the most widely used in biotechnology and,
recently, in in vivo imaging. 242,243 The luciferin/luciferase pairs emit at
relatively short wavelengths (for most luciferins between 480 and
560 nm); therefore, efforts have been made to obtain mutant luciferins or
use alternative substrates to shift the resulting bioluminescence toward