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of lanthanides is their long-lived fluorescence, usually in the millisecond
range, a lifetime at least 100,000-fold longer as compared to that of classical
fluorophores with typical lifetimes of upto 10 ns. Upon excitation, TR-
FRET donors can induce a delayed emission from the classic acceptor
fluorophores. This peculiarity enables the detection of a fluorescent signal
in a time-resolved manner, allowing the excitation and the detection
processes to be separated temporally (see
Fig. 7.1
). Usually, a 50-100
m
s
delay is applied between excitation and recording of the emission signal,
which rejects most of the background short-lived emission arising from
biological media, instrumentation, and microtiter plates, or by the chemical
compounds to be tested in HTS. This time-resolved detection is a first
major improvement over the actual fluorescence measurements in which
the fluorescence of some molecules often impairs their analysis in screening
assays.
2.3. Advantages of TR-FRET over classic FRET
Besides the main advantage of allowing time-resolved detection, TR-FRET
lanthanide donors also present peculiar emission spectra, allowing really high
signal-to-noise ratios. In fact, as for classic FRET, the energetic compatibil-
ity between the two fluorophores of the FRET pair is needed in TR-FRET,
which will be the case if the absorption spectrum of the acceptor overlaps the
emission spectrum of the donor. Lanthanide TR-FRET donors, especially
europium and terbium, present very large Stokes shifts, that is, the difference
in wavelength between positions of the band maxima of the absorption and
emission spectra of the fluorophore, and emit at several wavelengths with
sharp emission peaks (see
Fig. 7.1
). These specificities confer several advan-
tages. First is better spectral selectivity, as absorption and emission spectra of
the donor and acceptor fluorophores are well separated as compared to the
nonnegligible overlap of classic FRET or BRET pairs which results, for clas-
sic FRET, in the direct excitation of the acceptor by the excitation light and
thus further increases the background signal. Second, a variety of acceptors
can be used, thanks to the multiple emission peaks of the donors. In the
case of europium, the acceptors are preferably near-infrared fluorophores,
while terbium complexes offer more flexibility, as multiple acceptors evenly
disposed in the emission spectrumcanbe used (e.g., fluorescein-, rhodamine-,
or indocyanine-derived acceptors). Third, just like classic FRET, TR-FRET
is dependent on the distance between the fluorophore pair (
R
) relative to their
R
0
. The variety of donor-acceptor couples that can be used in TR-FRET
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