cell surface, but also free aptamer diffusing above the cell surface, will be seen. When a
portion of the aptamers in the confocal volume binds to the membrane receptors, the
bound aptamers are restricted to the cell surface and diffuse slower. Thus, the diffusion
time of the bound aptamers increases compared to the free ones. Diffusions of free
aptamers above the cell surface are described by the 3-D diffusion function, while
diffusions of bound aptamers on the cell membrane are represented by the 2-D diffusion
function. Thus, if the aptamer/receptor complex is stable on the time scale during its
transit through the detection region, then the overall autocorrelation function describing
the activities that occur in the confocal volume will represent a linear combination of the
autocorrelation functions of free and bound aptamers:
1 1 1 1
G(= (1-)- (3-1) 104
1+ 1 1+
free 2 bound
0 OZ Dfree
where N is the total absolute number of fluorescent molecules inside the focus,
TDfree is the diffusion time for the unbound labeled aptamer, and TDbound is the diffusion
time for the bound labeled aptamer. (1-r) is the fraction of the unbound aptamer
diffusing with TDfree, and r is the fraction of the bound aptamer diffusing with TDbound
Autocorrelation functions of FITC-labeled aptamers in solution and bound to the
cell membranes are respectively shown in Figure 3-2. Here we see that the binding of
aptamers to receptor results in an increase of the diffusion time, where TD = 0.827ms
(A), when compared to the free ones, where TD= 0.235ms (m), thus enabling FCS to