Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 465
Figure 13.32. Membrane fusion detection by RET. Top: Homotransfer
and self-quenching. Bottom: RET for a D–A pair.
ized around gramicidin then RET to the dansyl acceptor
would exceed the calculated values.
13.8.2. Membrane Fusion and Lipid Exchange
Energy transfer has been widely used to study fusion and/or
aggregation of membranes. These experiments are shown
schematically in Figure 13.32. Suppose a lipid vesicle contains
a high concentration of a fluorophore that undergoes
homo-RET (Figure 13.32). Initially the membrane will be
dimly fluorescent because of self-quenching (top). If this
vesicle fuses with another unlabeled vesicle the extent of
self-quenching will decrease and the emission intensity will
increase. An alternative approach is to use RET between a
donor and acceptor (bottom). In this case the vesicles
labeled with each type of fluorophore will appear with different
colors. Upon fusion the color will change. Depending
upon the concentrations of donor and acceptor in the
membranes, the extent of RET could be small, in which
case the color of the fused vesicles would be a simple mixture
of the two colors. If the probe concentration results in
efficient RET then the fused vesicles will have the color of
the acceptor.
As seen from Figure 13.28 the acceptor density does
not need to be large. Any process that dilutes the donor and
acceptors from the initially labeled vesicles will result in
less energy transfer and increased donor emission. For
example, if the D–A-labeled vesicles fuse with an unlabeled
vesicle, the acceptor becomes more dilute and the donor
intensity increases. Alternatively, the donor may display a
modest water solubility adequate to allow exchange
between vesicles. Then some of the donors will migrate to
the acceptor-free vesicles and again the donor fluorescence
will increase. It is also possible to trap a water-soluble fluorophore–quencher
pair inside the vesicles. Upon fusion
the quencher can be diluted and/or released. A wide variety
of different procedures have been proposed, 73–79 but most
rely on these simple proximity considerations.
RET has been used to obtain images of membranes as
they fuse together. Figure 13.33 shows images of giant unilamellar
vesicles (GUVs) that were labeled either with a
cyanine dye DiO (left, green) or a rhodamine dye Rh–PE
(right, orange). 80 The two types of vesicles contained oppositely
charged lipids, which resulted in rapid fusion. The
GUVs were brought together by electrophoretic migration
in a fluid channel. The images were obtained with a color
CCD camera, so they are real color images. Prior to contact
between the GUVs, the DiO-labeled vesicle is bright and
the Rh–PE-labeled vesicle dark. Rh–PE is dark because it
absorbs weakly at the excitation wavelength of near 450
nm. As the vesicles make contact and fuse, a bright orange
signal appears, which is due to Rh–PE. Eventually the
entire vesicle surface becomes orange. The green emission
from DiO is mostly quenched due to RET to Rh–PE.
13.9. EFFECT OF τ 2 ON RET
While the effect of κ 2 in energy transfer is frequently discussed,
81–82 there are relatively few experimental
results. 83–86 Most of the experimental results are measurements
with a single κ 2 value, rather than a systematic
dependence of k T (r) or κ 2 . However, the experimental
results show that the value of κ 2 does affect the rate of energy
transfer. Figure 13.34 shows the chemical structure of
two donor–acceptor pairs. The anthracene donor was linked
either in plane with the porphyrin acceptor (κ 2 = 4) or in the
axial position (κ 2 = 0) The transition moment of the
anthracene is along the short axis of the molecule. Emission
spectra of these D–A pairs were compared to a donor control
molecule. For the axial anthracene (κ 2 = 0) there is
almost no RET to the porphyrin. For the in-plane
anthracene with κ 2 = 4 there is almost no emission showing
RET is rapid for these colinear transitions. These results
show that orientation can have a dramatic effect on the RET
efficiency. However, these D–A pairs are rather rigidly
linked and a smaller effect is expected if the acceptor had
more mobility relative to the porphyrin ring.