Principles of Fluorescence Spectroscopy

820 FLUORESCENCE CORRELATION SPECTROSCOPY
Figure 24.31. Top: Autocorrelation curves for pure Cy5, pure FR662,
and a mixture. Bottom: G(τ) resolved from the mixture using the
time-resolved decays. Revised from [100].
24.10. DETECTION OF CONFORMATIONAL
DYNAMICS IN MACROMOLECULES
Since FCS can detect blinking and chemical reactions that
change the intensity, it seems natural to use FCS to study
the conformational dynamics of macromolecules. 102–108
Given the ms timescale of FCS, and the possibility of
increasing the diffusion time by increasing the size of the
volume, FCS should be able to detect conformational
changes that occur during the diffusion time. Since the
observed volume is limited by background emission, the
observed volume cannot be made too large, so the events
will probably need to occur on the microsecond timescale
or faster to be detectable by FCS. One example of measuring
macromolecular dynamics is shown in Figure 24.32.
This shows a molecular beacon that is opening and closing
with rate constants k 1 and k 2 . The open state is fluorescent
and the closed state quenched.
The correlation functions were measured for the beacon
G B (τ) and for a control oligonucleotide G C (τ) that did
not have the quencher (Figure 24.32, middle panel). While
Figure 24.32. Folding kinetics of a molecular beacon at 45°C.
Revised from [108].
the difference between G B (τ) and G C (τ) seems substantial,
the difference in amplitude may be the result of about 65%
the beacon being in the closed state at the experimental
temperature of 45EC, thus reducing the effective fluorophore
concentration. If the timescales are very different,
the overall correlation function is the product of the functions
due to the different processes. In this case the control
molecule reveals the portion of G(τ) due to translational diffusion:
The overall correlation function is given by
G B(τ) G C(τ) [ 1
GC(τ) 1 ( 1
N τ ) τD 1
1 p
p
τ
exp ( )] τR (24.42)
(24.43)