Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 497
Figure 14.26. Structure of chymotrypsin inhibitor 2 and single-molecule
RET efficiencies. Revised from [49].
14.6. TIME-RESOLVED RET IMAGING
RET is beginning to be used with fluorescence-lifetime
imaging microscopy (FLIM) to study association reactions
in cells. 50–56 This is a promising approach because within
reasonable limits the lifetimes can be independent of the
local probe concentration. Such measurements require spatially
resolved measurements of the donor intensity decays.
One approach is to use a streak camera that provides spatial
resolution along one axis and time resolution along another.
Such measurements are shown for Vero cells in Figure
14.27. The genes for CFP or YFP were fused with the gene
Figure 14.27. Intracellular CFP donor decays for RET between CFP
and YFP in Vero cells. CFP and YFP were linked to monomers of
thymidine kinase (TK). The transition from red to blue represents
decreasing intensity. Revised from [55].
for thymidine kinase (TK) from herpes simplex virus. 55
These two constructs were expressed in Vero cells. The TK
monomers form dimers in solution. The goal of the experiment
was to detect intracellular dimers. Figure 14.27 shows
streak-camera measurements of the CFP-TK donor in the
cells. The x-axis represents the boxed region across the
cells. The top panel shows the decay of the donor in the
absence of acceptor, and the middle panel shows the donor
decays for a cell transfected with the genes for both
CFP–TK and YFP–TK. The CFP donor decays move rapidly
in the cell transfected with CFP–TK and YFP–TK than in
the cell transfected with only CFP–TK. The lower panel
shows the total counts along the x-axis. The shape of the
decay for the cell with both D and A suggests that a fraction
of the CFP–TK is associated with acceptor and a fraction of
CFP–TK probably does not have a nearby acceptor. This
result is expected because TK forms a homodimer so the
cell will contain D–D, A–A, as well as D–A pairs.