Biomedical Engineering Reference
In-Depth Information
spectroscopic experiments that can be carried out to determine information on parameters
such as FRET distance, orientation factor, and the distance between donor and acceptor. The
donor-acceptor distance can be determined by both steady state and time-resolved fluores-
cence measurements (17). The steady-state measurement of three different parameters can be
taken to determine
r
: decrease in donor fluorescence, comparison of the absorption spectrum
and the excitation spectrum (through observation of the acceptor fluorescence), or enhance-
ment of acceptor fluorescence (17). In the time-resolved regime, decay of donor fluorescence
and increase in the acceptor fluorescence (if the acceptor fluoresces in the wavelength region
capable of being detected by the instrumentation used for the experiment) can be used to
determine a value of
r
(17). One limitation of using these methods is that the distance between
donor and acceptor is assumed to be constant; however, if the donor and acceptor are linked
by a flexible chain such as a nucleic acid strand, the donor-acceptor distance is not always
constant. Similarly, if the donor and acceptor are linked to two different nucleic acid strands,
there is still a degree of uncertainty in the location of the donor and acceptor relative to one
another. In this case, the method used for the estimation of a distribution of donor-acceptor
distances is based on the measurement of donor fluorescence decay and the shape the decay
profile takes as a result of these variable interactions between the donor and the acceptor (17).
Time-resolved donor fluorescence decay gives the best approximation to the orientation and
distance between the donor-acceptor pair; however, at best, this is only an average distance
where the distance distribution has contributions from both the distance and the orientation
parameters (17).
Viscosity of the medium that the donor-acceptor pair experiences also affects the dynam-
ics of energy transfer because of the dependence of diffusion on viscosity (17). The distance
that the donor and acceptor diffuse toward each other during the excited-state lifetime of the
donor is dependent upon viscosity (17). If the donor and acceptor are unable to diffuse a sig-
nificant distance toward one another during the excited-state lifetime of the donor, then a
static limit exists between the donor and the acceptor. As a consequence, two modes of energy
transfer are possible: the Perrin model of the sphere of effective quenching can approximate
the energy transfer process, and if the distance between donor and acceptor is less than 2
R
0
,
then the energy transfer occurs via Förster transition dipole interactions (17). If the mean dis-
tance the donor and acceptor diffuse relative to each other is larger than the mean separation
distance,
r
, during the excited-state lifetime of the donor species, then once again two modes
of energy transfer are possible (17). If energy transfer occurs via a collisional process, then the
Stern
-
Volmer model for kinetics is used to define the donor-acceptor system; if the Förster
mechanism is considered, then a rapid diffusion limit exists (17). The energy transfer from the
donor to acceptor is transferred to acceptors that lie in the <2
R
0
distance range, as well as any
acceptors that happen to diffuse to within that distance during the excited-state lifetime of the
donor species (17). If the mean distance diffused by the donor and acceptor relative to each
other during the excited-state lifetime is approximately equal to the distance
r
between the
donor and the acceptor, then a very complex case is encountered and requires an intricate set
of mathematical expressions to approximate values for FRET (17). Restrictions in terms of ori-
entation geometry also bring about deviations from the “ideal” model of Förster decay (17).
2.2.4
Signal-to-Noise and the Practical Issues of Single Molecule Detection
Perhaps the main practical concern encountered in the use of fluorescence techniques is the
improvement of the signal-to-noise ratio. This problem becomes a fundamental obstacle that
must be addressed in the case of single molecule detection. Raman and Rayleigh scattering
can produce significant background contributions, as can fluorescence from the environ-
ment surrounding the target molecule (e.g., the solid substrate in the case of an immobilized
single molecule), and fluorescence from the detection optics. To ameliorate these problems,
