Biomedical Engineering Reference
In-Depth Information
2.2.3.6 Solvent Viscosity/Twist Angle: (Example—the Cyanine Dyes)
Cyanine molecules possess two resonance structures where there is a charged and an
uncharged nitrogen atom in each resonance structure. This results in a symmetry of charge
distribution when both of the isomers are considered (17). This leads to complexity in terms
of the photophysics of relaxation. The fluorescence quantum yield and decay are depend-
ent upon both twist angle and solvent viscosity (17). The nonradiative decay rate depends
on twist angle, and consequently quantum yield depends on solvent viscosity/rigidity of
surrounding media (17). For example, thiazole orange, an unsymmetric cyanine dye, has a
double bond structure that allows intramolecular rotation to occur. It also contains two
nitrogen atoms that create charge symmetry due to resonance. Thus, thiazole orange is
weakly fluorescent in solution. However, it is highly fluorescent in viscous or rigid media,
which makes it an excellent choice as an intercalator indicative of DNA hybridization.
Bridging rotational bonds between two ring structures with an oxygen atom can also
restrict the bridging bond rotations (17). Thus, reduction in rotation is achieved, which is
the main mode of reducing bond rotation between bridging bonds of the substituted
phenyl rings in rhodamine dyes (17). This allows rhodamine dyes to be fluorescent in
polar media and exhibit high quantum yields in polar solvents as a result (17). The fluo-
rescence emission from fluorophores is therefore shown to be very sensitive to the
structure of fluorophores as well as the immediate environment. This allows them to be
excellent candidates for use as fluorescent probes in applications such as detection of
nucleic acid hybridization. The characteristics of fluorescence (spectrum, quantum yield,
lifetime) can provide a wealth of information about the environment close to the
fluorophore, and thus fluorescence spectroscopy becomes a powerful investigative tool
that provides spatial and temporal information with excellent sensitivity.
2.2.3.7 Quenching
Since quantum yield is essentially a measure of the fraction of photons emitted to the
number of photons absorbed, a theoretical upper limit for
would be equal to unity. This
value is never achieved for molecules due to nonradiative energy losses as well as a
phenomenon that contribute to fluorescence quenching. The main photophysical
processes that contribute to fluorescence quenching are the following: collisions with
atoms/other molecules; electron transfer, excimer formation, exciplex formation; proton
transfer, and energy transfer (17). Fluorescence energy transfer is an important mode of
fluorescence quenching and is discussed in an individual section. Fluorescence quenching
can also be an effective way to interrogate the environment near a fluorophore both quan-
titatively and qualitatively. Several molecular deactivation processes that lead to the
quenching of fluorescence are outlined below.
2.2.3.8 Dynamic Quenching: (Example—Stern-Volmer Relation)
In the initial treatment of fluorescence quenching, the quenching phenomenon is assumed
to be time-independent and, in essence, does not consider the role of diffusion-limited
processes. This leads to a fluorescence decay, which is a single exponential profile, and the
Stern
-
Volmer relation can then be represented using the following equation:
I
I
[2.5]
0
0
1
k
Q1
K
Q
q
0
SV
where
I
0
and
I
are the fluorescence intensities in the absence and presence of quencher,
respectively,
0
and
are the quantum yield terms in the absence and presence of
