engineering feature that makes FCS a highly sensitive biophysical tool for studying
molecular interactions, especially in live cells.
Single-Component Diffusion and Multi-Component Diffusion
Single-component diffusion generally describes molecular diffusion of one
specific type of molecule in its specific state or conformation inside the laser focus
volume. The approach to obtain the molecular diffusion information is similar to that
described above. The dimensions of the focus volume are first obtained from a free dye
calibration. Then with the fixed wxy and w, in Equation (2-2), the diffusion time r- of the
molecule in the single-component system can be obtained.
Besides single-component diffusion, the large diffusion time range accessible by
FCS makes it possible to analyze the superposition of various diffusion (and/or blinking)
processes that take place on different time scales in a single FCS measurement. Their
respective time scales can be revealed by fitting the autocorrelation curve to a multi-
component diffusion model. For instance, the model for multi-components XA, XB, Xc, ..
with diffusion times rA, TB, zC, ..., reads
G3D()= r + r r +... B
N 1+ 1+ I+ 1 + I+- I+
TA 2 TB C 2 C C 2
S\TA B C
(2-9)
where rA, rB, rc, ..., represent the fractions of components XA, XB, Xc, ..., respectively,
and are obtained from G(T) curve fitting. wxy and w, represent the geometry of the focus
volume shared by components XA, XB, Xc, ....