Biology Reference
In-Depth Information
Application of FRET and Microscopy
to create the so-called monomeric versions of GFP derivatives, which more or less
sufficiently suppress the native dimerization tendency.
Since the dimensions of GFPs are already in the order of
R
0
, proper linker design is
very crucial. Hence, appropriate linker length and steric conformation (includes dis-
tance and orientation) flexibility is necessary. At the same time, the functionality of
receptor is essential, in particular if the analysis of downstream signaling is of interest.
To quantify FRET, not only high FRET efficiency but also sufficient brightness
of FPs is important. High quantum yield and superior excitation coefficients of FPs
can be achieved by using new optimized CFP and YFP derivatives. High quantum
yield of donor and acceptor fluorophores improves quality of FRET investigations by
increasing signal-to-noise ratio, which is important for pixel-based FRET quantifi-
cation in live cells. From the intensity level of the signal, one can estimate the ex-
pression level of receptors and also exclude saturation artifacts.
14.1.3
Principle behind lux-FRET quantification and its outcome
There are several methods available to quantify FRET. Applying fluorescence
lifetime-based methods, the FRET efficiency
E
can be determined directly. How-
ever, biologically relevant in most types of experimental designs is the fraction of
molecules present in FRET state. From fluorescence lifetime techniques, like
time-correlated single-photon counting, the fraction of FRET complexes scaled
by total donor concentration
f
D
DA
D
t
, where
D
is donor
and
A
is acceptor, can be obtained from the fractional amplitudes of the multiexpo-
nential decay
A
t
DA
and
A
t
D
(
Zeug et al., 2012
). For reliable results, a high number of
photons are required, which limits the spatiotemporal resolution and makes it rarely
suitable for live-cell imaging. Moreover, the fraction of FRET complexes scaled by
total acceptor concentration
f
A
½
=
½¼
A
t
DA
=
ð
A
t
DA
þ
A
t
D
Þ
[
DA
]/[
A
t
] cannot be directly obtained from fluores-
cence lifetime. Therefore, to study protein-protein interaction, we developed lux-
FRET (
Wlodarczyk et al., 2008
); for an overview of spectral FRET approaches,
see also
Zeug et al. (2012)
). Lux-FRET is a spectral quantitative FRET approach
based on two sequential excitations and linear unmixing of fluorescence emission
by spectral references of donor
F
i
,ref
(l) and acceptor
F
i
,ref
(l) at the corresponding
excitation wavelength
i
:
F
i
ðÞ¼
d
i
F
i;
ref
D
ðÞþ
a
i
F
i;
ref
A
(14.2)
From the contributions d
i
and a
i
from donor and acceptor emission, respectively, the
apparent FRET efficiencies
Ef
D
and
Ef
A
and the donor mole fraction
x
D
can be
obtained by
ðÞ
E
DA
½
Da
Ef
D
½
¼
(14.3)
D
t
r
d
1
D
þ Da
E
DA
½
Da
Ef
A
½
¼
R
TC
(14.4)
A
t
a
2
r
ex
;
1
a
1
r
ex
;
2
