Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 407
Figure 11.34. Correlation time images of R6G in various solvents (top) and histograms of the recovered correlation times (bottom). The upper three
images are R6G in methanol. Revised from [105].
nent to about 6 ns. The polarized intensity decays are visually
seen to be multi-exponential as predicted by eqs. 11.1
and 11.2.
The time-dependent decays were used to create FLIM
images of the samples (Figure 11.33, left). The lifetimes at
each pixel in the images are apparent lifetimes that are
some weighted averages of the multi-exponential decays. In
methanol the apparent lifetimes are the same regardless of
the polarization, as expected based on the short correlation
time. In ethylene glycol the apparent lifetime of the parallel
component is shorter than the perpendicular component.
The parallel component has a shorter apparent lifetime
because the transition moments are rotating away from the
orientation of the observation polarizer. These results show
that the gated polarized intensity images contain information
in the correlation times of the samples.
There can be some confusion about the difference in
apparent lifetime with horizontal excitation (Figure 11.33,
lower left). With the usual right angle geometry horizontal
excitation results in the same signals in the parallel and perpendicular
channels (Section 10.4). The geometry is different
for the instrument in Figure 11.32 where the emission is
observed along the same axis as the excitation. For this
geometry rotation of the excitation polarizer reverses the
signals in the two polarized observation channels.
The polarized time-dependent intensities can be used
to calculate the correlation times (Figure 11.34). The color
of the images reveals the longer correlation times of R6G in