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concentrations and are prone to complications due to channel cross-talk.
Dequenching of donor fluorescence through the photobleaching of the
acceptor population provides a more quantitative approach to FRET analysis
as pre-bleach acceptor intensities can be compared with post-bleach values.
Therefore, where possible, it is desirable to combine these two techniques as
each provides an independent evaluation of the FRET process. An excellent
review and comparison of these and other techniques is provided elsewhere
(Wouters et al., 2001).
FLIM provides one method that enables a quantitative measure of FRET.
The fluorescence lifetime of a fluorophore is defined as the average duration
the molecule remains in the excited state following photon absorption
(Bastiaens and Squire, 1999). The lifetime of a donor fluorophore is a useful
parameter to measure because it is sensitive to local environmental changes
such as FRET interactions whilst insensitive to fluorophore concentration, a
factor that can complicate measurements based on absolute intensities. Two
techniques have been developed that enable fluorescence lifetimes to be
measured: frequency domain and time domain FLIM. In frequency domain
FLIM the continuous hi-frequency modulation of the excitation intensity is
used to modulate the intensity of fluorescence emission. The frequency of
intensity modulation is the same for both excitation and emission wavelengths
but differences exist between their phase and relative modulation depth. These
differences are determined by the fluorescence lifetime of the fluorophore and
can be used as independent parameters for its derivation. By modulating the
sensitivity/gain of the detector at the same frequency as the light intensity
modulation, phase-sensitive imaging is enabled allowing the differences in
phase and modulation depth to be calculated (Bastiaens and Squire, 1999;
Squire and Bastiaens, 1999). Alternatively, the time domain technique uses
pulsed excitation of the sample. The pulsed excitation of a population of a
single species of fluorophore results in fluorescence emission that decays
exponentially with time. The lifetime of the fluorophore is derived from this
decay curve and is defined as 1/e, which is the time taken for the intensity of
fluorescence emission to decay to approximately 37% of its initial value
(Bastiaens and Squire, 1999; Benny Lee et al., 2001).
One of the most powerful features of FRET/FLIM analysis is that it
enables biochemical processes to be imaged in individual living cells. Any
spatial constraints that affect the subcellular localization of a protein should
apply and therefore the technique is ideal for the study of spatio-temporal
signalling relationships. Furthermore, single cell imaging provides the
opportunity to study the signalling heterogeneity that can exist within a
population of cells, something that is impossible to achieve using conventional
biochemical techniques.
The use of FLIM in the analysis of ErbB1 receptor activation dynamics
provides an elegant example of how microscopy-based analysis of biochemical
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