Biomedical Engineering Reference
In-Depth Information
collisional processes between molecules, solvent effects and reactions, which occur while
the molecule is in the excited state (17). Stokes' shift measurements can reveal important
information about a molecule. For example, when the excited state dipole moment is
higher than the ground state dipole moment, the Stokes' shift increases as a function of
increasing solvent polarity (17).
2.2.2
Measurement of Fluorescence
Fluorescence intensity is defined by an extension of the Beer-Lambert law:
bC
[2.1]
F
I
0
(1
10
)
is the quantum yield,
K
is the instrument response coefficient,
I
0
is the intensity of the excitation radiation,
where
F
is fluorescence,
is the molar absorptivity of the fluorophore,
b
is the pathlength, and
C
is the concentration of fluorescent species. If the concentration
of the species is relatively low (below 0.01 M), then the equation can be approximated as
[2.2]
F
2.303
KI
bC
0
When considering the use of fluorophores as an analytical tool, the quantum yield of flu-
orescence must be taken into account as it directly relates to the intensity of the fluores-
cence signal generated from the population of fluorophores under interrogation. The
quantum yield of fluorescence is defined by:
k
r
[2.3]
k
k
r
nr
where
k
r
represents the decay lifetime (rate) of the radiative processes and
k
nr
represents
the decay lifetime of the nonradiative processes. Upon inspection of Equation 2.3, as the
number of nonradiative processes and their lifetimes decrease, the quantum yield
approaches unity and as such, the quantum yield represents the fraction of excited fluo-
rophores that relax by fluorescence (radiative) emission.
The quantum yield of fluorescence is related to the lifetime of the excited state by the
following relationship:
k
[2.4]
k
r
kk
F
rs
r
nr
s
represents the excited state lifetime or fluorescence decay time of the singlet state.
This parameter defines the time window in which dynamic processes can be observed
(17). Several parameters influence quantum yield, and hence lifetime.
where
2.2.3
Parameters Affecting Fluorescence
2.2.3.1 Molecular Structure: (Example—Degree of Conjugation)
A significant number of fluorophores are aromatic. The
conjugation of aromatics leads
to a shift of the fluorescence excitation and emission maxima to longer wavelengths and
results in an increase in the fluorescence quantum yield as the degree of conjugation
increases (17).
