Figure 2.

Single-molecule kinetics in living cells. (A) Association and dissociation rate constants can be determined by statistical analysis of the single-molecule off-time and on-time measurements, respectively. The cartoon shows a simple two-state reaction. (B) Cluster size distribution can be determined from a histogram of single-molecule spot intensities. Here, cells were incubated with various concentrations of fluorescent EGF (TMR-EGF) for 20 s (left). The distributions (right) were fitted by the sum of four Gaussian functions (dashed lines). Gaussian distribution of the fluorescence intensity was confirmed from the single-molecule fluorescence intensity distribution of TMR-EGF on the glass surface. The fluorescence intensity was normalized with respect to that of a single molecule. (C) Using data from panel B and reaction models (left), the association rate constants between EGF and its receptor, and the ratio between receptor monomers and pre-dimers have been determined (right). In this case, the receptors are known to form monomers and pre-dimers. The total number of receptor per cell is also known to be 5.5 × 10⁴/cell (Berkers et al, 1991). See Supplementary information for details of the analysis.