Biology Reference
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The resolution of multiple t
i
becomes increasingly difficult as they get
more closely spaced. The statistically significant resolution of close t
i
re-
quires a high signal-to-noise ratio and a large number of collected photons.
The calculation of the energy transfer efficiency from the donor fluores-
cence lifetime in the presence and absence of the acceptor (Eq.
5.11
)as-
sumes that, under the experimental conditions, the donor decays
according to a single-exponential model. If the donor displays a multi-
exponential decay, t
i
values can be used to calculate the average lifetime
h
t
i
. It is defined as the average time that the fluorophore remains in the
excited state and is defined as
ð
1
tI t
ðÞ
d
t
X
ð
1
0
hi¼
d
t
¼
f
i
t
i
½
5
:
12
It
ðÞ
i
0
In order to simplify data representation, mean lifetime t
mean
has been
largely used in FRET experiments.
8-11
It is given by
ð
It
X
i
a
i
t
i
t
mean
¼
¼
ðÞ
d
t
½
5
:
13
3. FRET MEASUREMENT
3.1. Introduction
Among the different previously described nonradiative deactivation pro-
cesses (
K
nr
), the energy transfer between dipoles was first described by
F¨rster in 1926 and Perrin in 1932. F¨rster resonance energy transfer or FRET
is a physical process in which the energy of a chromophore (called “donor”
[D]) in its excited state is transferred nonradiatively to a neighboring
chromophore (called “acceptor” [A]) while in its ground state.
5
This phys-
ical process has been often applied experimentally for investigating molec-
ular interactions at distances beyond the diffraction-limited resolution (for
review see Refs.
5,7
). For instance, FRET measurements (generally used
in spectroscopy and microscopy) allow the investigation of the formation
of protein complexes in living cells and tissues, as well as the
conformational changes of single proteins such as biosensors (for review
see Refs.
6,7,12
).
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