Biomedical Engineering Reference
In-Depth Information
made. The resolution of a biexponential FRET system relies in part on the
correct selection of fluorophore, particularly for the donor.
Many FPs have been shown to have complex fluorescence decays, often
biexponential or even more complex, presumably due to protonation or some
internal relaxation process, even in a non-FRET system. An example of this
is ECFP (enhanced cyan fluorescent protein). This is commonly used as a
FRET donor, because of its high quantum yield, long emission tail, and rel-
atively blue emission. However, several fluorescence lifetime studies have re-
vealed ECFP to have a biexponential lifetime [41, 42], making it di
cult to
resolve the interacting fraction from a noninteracting fraction in a FRET sys-
tem (using FLIM); to do this, four lifetimes would need to be fitted to the
data (i.e., two for the non-FRET system, and two for the FRET system), and
it is beyond most FLIM measurements to acquire accurate enough data in
a practical time period. However, there are FPs useful as FRET donors in
FLIM measurements. EGFP (enhanced GFP) has a high quantum yield and
a monoexponential decay in a non-FRET system, and has been shown to be
a good donor for red FPs, such as dsRed and mRFP [monomeric RFP (red
fluorescent protein)] [43] (Fig. 10.12). One advantage of EGFP for many cell
top membrane
ventral membrane
Autocorrelation functions
Fluorescence Intensity images
1.00
high
0.75
0.50
ventral
membrane
0.25
top
membrane
low
TCSPC - FLIM images
0.00
10
FRET
0 +/− 8%
0.0001
0.001
0.01
0.1
1
tau (s)
FRET
18 +/− 8%
Fig. 10.12.
Study of molecular association and diffusion at the cell membrane of
the active GPI-anchored uPAR, fused to EGFP and transfected in human kidney
cells, HEK293.
Top panel s
: two-photon images of live cells showing the distribution
of the receptor at the ventral and topside of the cell membrane; Representative
ACF acquired in two regions at the top and ventral membrane (+) showing that the
receptor has longer diffusion time at the cell adhesion site (about 0.9 and 0.1
µ
m
2
/s
respectively).
Lower panels
: FLIM analysis of cells cotransfected with the GFP-
mRFP1 receptor pair showing that the regions where the receptor is slow diffusing
are also regions of high FRET e
ciency
