Biomedical Engineering Reference
In-Depth Information
F
F
1
[2.16]
0
1
K
s
Q
If fluorescence intensity is proportional to the concentration of the fluorophores, then this
equation can be inverted and expressed in terms of intensity:
I
I
[2.17]
0
1
K
s
Q
Static and dynamic quenching can be encountered simultaneously as well. Cases where
there are multiple interactions lead to deviations in the linearity of Stern
-
Volmer plots for
the F and Q systems involved as well as complexities in combining mathematical rela-
tionships of both dynamic and static expressions.
In addition to the static and dynamic quenching models, several other modes of
fluorescence quenching exist such as photoinduced electron transfer, excimer/exciplex
formation, and photoinduced proton transfer. Transfer of energy from a donor chro-
mophore to an acceptor species is also a mode of fluorescence quenching. This transfer is
referred to as fluorescence resonance energy transfer (FRET).
2.2.3.12 Fluorescence Resonance Energy Transfer
When FRET occurs, the relaxation energy of the donor species is absorbed by the acceptor
such that the fluorescence emission of the donor species is effectively quenched by the accep-
tor provided that there is sufficient spectral overlap between the fluorescence emission and
the acceptor absorbance wavelengths. In this donor-acceptor relationship, the fluorescence
emission of the donor is quenched by the acceptor. The acceptor, which absorbs the relaxation
energy emitted by the donor species, then undergoes relaxation processes, and this emission
of radiation can either be in the visible range of the electromagnetic spectrum or lie beyond
the detection wavelength range for typical fluorescence detection instrumentation.
Energy transfer from a donor to an acceptor where the donor and the acceptor are two
different molecules can be represented by the simple equation:
[2.18]
DADA
The energy transfer between D and A can be either radiative or nonradiative (17). FRET
follows the nonradiative transfer mode and will be discussed exclusively below.
In nonradiative transfer, there is no emission of photons from the donor at distances
shorter than the wavelength of excitation (17). This quantum mechanical photophysical
process occurs when there is sufficient spectral overlap between the emission spectrum of
the donor and the absorption spectrum of the acceptor. This overlap ensures that there are
a sufficient number of vibronic transitions which are equal in energy in both the donor and
the acceptor species such that the transitions are in resonance (17).
The energy transfer between the donor and the acceptor is due to several interactions
between the two species. The interactions can be due to either short-range intermolecular
orbital overlap or long-range coulombic interactions (17). Long-range coulombic interac-
tions are long-range dipole-dipole interactions, and are also referred to as the Förster
mechanism of interaction (17). This coulombic term refers to the energy transfer process
where the excited electron occupying the LUMO of the donor relaxes and an electron in
the HOMO of the acceptor are excited to the LUMO of the acceptor simultaneously (17).
In this case, long-range interaction occurs over distances up to 8-10 nm (in comparison to
short-range interactions which are a few tens of Angstroms) (17).
