Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 815
ity in membranes. When performing FRAP a laser is
focused on the region of interest in the membrane. 74–76 The
laser is transiently brought to high intensity to photobleach
the fluorophores in the focal region. The laser intensity is
then decreased to allow continuous monitoring of the emission
from this same region of the membrane. The intensity
increases as the unbleached probes diffuse into the bleached
area, and the rate of recovery is used to determine the diffusion
coefficients. In this FRAP measurement the experimental
system was synchronized with the bleaching pulse
and the system studied as it returned to equilibrium. The
system is not stationary during the experiment. In contrast
to FRAP, FCS measurements are performed under stationary
conditions. The system is hopefully not perturbed by the
illumination needed for FCS. Information about mobility in
the membrane is obtained by diffusion of the probe molecules
under equilibrium conditions.
24.6. EFFECTS OF INTERSYSTEM CROSSING
In the preceding sections we described the effects of translational
diffusion on the autocorrelation functions of
labeled molecules. Diffusion is just one of several mechanisms
that can cause intensity fluctuations, and such
processes can be studied by FCS. Because of the high illumination
intensities in FCS, intersystem crossing from the
first excited singlet state (S 1 ) to the triplet state (T) is frequently
observed. A Jablonski diagram for intersystem
crossing is shown in Figure 24.23. The excitation intensities
used in FCS can result in a significant fraction of the fluorophores
in the triplet state. The fluorophores in the triplet
state are not observed, resulting in an apparent decrease in
Figure 24.23. Jablonski diagram with intersystem crossing from the
singlet (S) to the triplet (T) state. k 10 is the sum of the radiative and
non-radiative decay rates.
Figure 24.24. Effect of illumination intensity, iodide, and oxygen on
the normalized autocorrelation function of rhodamine 6G in water.
Revised and reprinted with permission from [78]. Copyright © 1995,
American Chemical Society.
the number of fluorophores in the effective volume. If the
fluorophores do not return to S 0 within the diffusion time
then only the amplitude of the correlation function will be
changed. If the triplet fluorophores can return to S 0 within
the diffusion time then this is a mechanism that can cause
fluctuations or blinking of the fluorophores, 77–80 as is also
observed in single-molecule experiments.
Prior to describing the theory for blinking in FCS it is
informative to examine some examples. The top panel in
Figure 24.24 shows autocorrelation functions for rhodamine
6G (R6G) in water, using different illumination
intensities. At low incident power of 48 µW, G(τ) appears
normal with a diffusion time near 0.03 ms (top panel). As
the incident power increases a new component appears in