CHAPTER 4
UPGRADING FCS TO DUAL COLOR-FLUORESCENCE CROSS CORRELATION
SPECTROSCOPY (FCCS) FOR WIDER APPLICATION TO MOLECULAR
INTERACTION STUDIES
Limitations of FCS Autocorrelation Analysis for Molecular Interaction Studies
Although the autocorrelation analysis in FCS appears to be a sensitive tool for
molecular interaction studies, these can be achieved only if binding results in a
measurable change in mobility (or other quantity accessible from single-color data).
Examples of applicable systems include some membrane studies where the ligand
undergoes a significant reduction in mobility when bound to the plasma membrane of a
cell, as discussed in Chapter 3, or to an artificial membrane that is (quasi-) planar. A
number of binding experiments have also been performed in vitro, based on the change
in diffusion time.129-133
As discussed in Chapter 2, from Equation (2-4), it is clear that the mean diffusion
time TD is related to the reverse reciprocal of the diffusion coefficient D:
co 1
S-- > zD oc
4D D (4-1)
According to the Einstein-Stokes Equation, the diffusion coefficient D is inversely
proportional to the radius of the molecule:
kT 1
D=- -Doc--
6rr/R R (4-2)
in which k is Boltzmann's constant;
T is the absolute temperature;
/7 is viscosity;