Time-Resolved Förster Resonance Energy Transfer-Based Technologies to Investigate G Protein-Coupled Receptor Machinery: High-Throughput Screening Assays and Future Development - Progress in Molecular Biology and Translational Science - page 280

A Temporal selectivity

FRET

Fluorescence
intensity

Bound
fluorophores

Free
acceptor

Medium
fluorescence

Free
donor

0 50 400 Time ( m s)

Delay Measure

Laser pulse

B

Spectral selectivity

Excitation Green FRET Red FRET

1.0

0.5

0.0

300 400 500 600 700

l (nm)

Absorbance Fluorescence

SNAP-Lumi4Tb
SNAP-Green
SNAP-Red

Figure 7.1 Specificities of lanthanides as FRET donors. (A) Time-resolved detection of
fluorescent/FRET signals. Thanks to long fluorescence lifetime of the lanthanides used
in TR-FRET, a delay is applied between excitation by a laser pulse and recordings of the
fluorescence/FRET signals, allowing suppression of all interfering short-lifetime emis-
sions, a phenomenon known as “temporal selectivity.” (B) Absorption and emission
spectra of SNAP-Lumi4Tb, a donor used in TR-FRET (purple), and SNAP-Green (green)
and SNAP-Red (Red), two acceptors compatible with the donor. Characteristics of
SNAP-Lumi4Tb spectra allow to transfer on several acceptor fluorophores and
induce a high spectral selectivity between the donor fluorescence emission at
620 nm and the acceptor

fluorescence emissions at 520 nm (SNAP-Green) and

665 nm (SNAP-Red).