Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 817
Figure 24.25. Normalized autocorrelation function for EGFP at various
pH values. Revised from [86].
which is assigned to pH-dependent protonation of the tyrosine
residue. The rate of protonation increases at lower pH
values, resulting in the increased amplitude at short times.
The proton may come from the bulk solution or from the
protein itself.
24.8. FLUORESCENCE INTENSITY
DISTRIBUTION ANALYSIS
In the preceding sections we considered processes that
result in intensity and/or concentration fluctuations in the
observed volume. The relative contributions of different
brightness fluorophores to the correlation function was
given in eq. 24.19, but we did not describe any approach to
resolve the different fluorophores from each other. The
presence of different brightness fluorophores changes the
apparent number of observed molecules (eq. 24.20), but the
information about their individual brightness values is lost
during collection of G(τ), as can be seen by examining eq.
24.10. The amplitude of the correlation function is due to
the same fluorophore emitting more than a single photon
during the binning time. During a particular time interval
the signal from the same dim fluorophores correlate with
each other, as will the signal from the bright fluorophores.
When the diffusion coefficients are the same the brightness
of each fluorophore cannot be resolved and the correlation
function will have the usual shape for a single diffusion
species.
Figure 24.26. Comparison of correlation function and photon-count
histogram for a mixture of dim and bright particles.
Suppose the sample contains two types of molecules
with the same diffusion coefficient (Figure 24.26). This
could be the same protein labeled with one or several fluorophores.
If G(τ) were measured for this mixture one would
see the usual correlation function for a single diffusion
coefficient (lower left). Suppose now that the entire time
course of intensities was available instead of the correlation
functions. There would be lower and higher intensity fluctuations
due to the dimmer and brighter particles, respectively.
One can count the number of times a fluctuation has
a dim or bright amplitude, and create a histogram of the
results (lower right). For the mixture there will be two populations
of fluorophores, which will be seen from the intensity
distributions. If the particles display very different
brightness it is possible to count the number of times dim
and bright particles pass through the observed volume. One
example is shown in Figure 24.27 for coumarin-labeled
Figure 24.27. Photon-count histogram for a mixture of 0.05 and 0.115
µm coumarin-labeled beads. Revised from [87].