Principles of Fluorescence Spectroscopy

814 FLUORESCENCE CORRELATION SPECTROSCOPY
Figure 24.22. FCS studies of insulin C-peptide binding to human renal tubular cells using rhodamine-labeled (Rh) C-peptide. The dashed lines show
G(τ) and P(τ) after addition of a 1000-fold excess of unlabeled C-peptide. Revised from [71].
G(τ) 1
N ∞
0
P(τ D)
(1 τ/τ D) dτ D
(24.36)
In the absence of the receptor the diffusion time is near 0.5
ms (lower left panel). In the presence of the receptor there
is a dramatic increase in τ D to 100 ms, with some fraction
of the peptide diffusing more rapidly (lower right panel).
There is nearly complete reversal of binding upon addition
of an excess of unlabeled C-peptide, showing the specificity
of C-peptide binding (dashed line, lower left).
FCS measurements of membranes provides information
that is different from fluorescence measurements in
bulk solution or fluorescence microscopy. It would not be
possible to detect lateral diffusion of the insulin receptor
using steady-state measurements because the nanosecond
decay times are short relative to the long times needed for
diffusion. Receptor diffusion would also be undetectable by
fluorescence microscopy because the system is roughly stationary.
The receptors are diffusing but the average distribution
is constant. FCS can detect receptor motion because
diffusion affects the number of fluorophores in the observed
volume, even under conditions when the average distribution
of receptors is not changing. The results shown in Figure
24.22 also indicate that the rate at which labeled C-peptide
dissociates from its receptor is slow relative to the
longest time, near one second, in the autocorrelation function.
It is instructive to compare FCS and fluorescence
recovery after photobleaching (FRAP) for studies of mobil-