images for measuring the donor quenching or donor lifetime for estimating
energy transfer efﬁciency (E %) [86, 87]. The ﬁgure illustrates images comparing
C-FRET, 2p-FRET, and FLIM-FRET. C-FRET and 2p-FRET images of the
quenched donor (a, c) and the PFRET images (b, d) are shown. The respective
efﬁciency (E ) and the distance (r) are shown in the table below the ﬁgure.
The distance between donor and acceptor molecules appears to be higher for
2p-FRET than for C-FRET. This may be due to the difference in methodology
of acquisition of photons. Both C-FRET and 2p-FRET signals were collected in
the same cell and optics using the Biorad Radiance2100 confocal/multiphoton
microscopy system. For the same cell, the donor lifetime images were acquired
in the absence (e) and the presence (f ) of acceptor. As stated in the text, the
natural lifetime of the donor (2.62 ns) was reduced to 1.9 ns (mean value) due to
FRET. Lifetime measurements are the accurate values of the distance distribu-
tion of the dimerization of C/EBPD244 protein molecules in mouse pituitary
GHFT1-5 cell nucleus .
In summary, FRET-FLIM is an important technique for investigating a variety
of phenomena that produce changes in molecular proximity and for monitoring
intermolecular interactions and localization of proteins in cells and tissues .
12.7.7 Fluorescence Recovery After
Fluorescence recovery after photobleaching is used for studying the dynamic
behavior of labeled molecules, speciﬁcally the behavior of proteins in living cells.
The process involves photobleaching a region of interest, thereby allowing the
temporal study of the consequent ﬂuorescent recovery in that bleached region as
a result of the movement of nonbleached ﬂuorescent molecules from the sur-
rounding area. The extent to which this recovery occurs and the speed at which it
occurs are measures for the fraction of mobile molecules and the speed at which
they move, respectively . The basic FRAP experiment is straightforward.
First, a region of limited dimensions within a larger volume is illuminated with
a short pulse of an intense laser beam at the excitation wavelength of the dye to
be bleached. Subsequently the molecules in the exposed region are no longer
ﬂuorescent. If the target labeled molecule is ﬁxed, the region will remain dark.
However, if the target molecules are mobile, they diffuse, with new ﬂuorescent
molecules from the surrounding unbleached regions moving into the bleached
region and mixing with the bleached molecules. This leads to a continuous
increase of ﬂuorescence in the bleached region until the bleached and new
ﬂuorescent molecules have been completely redistributed over the entire volume.
If the bleached area is relatively large compared to the total volume in which
the target molecules reside, the ﬁnal recovery of ﬂuorescence will be less than the
prebleaching level. This process can be followed on a microscope by visualizing
12 Fluorescence Imaging