Fig. 2. Tracking individual eYFP-H-Ras(wt) molecules in the plasma membrane of live cells. (A) Image showing three single-molecule signals of eYFP-H-Ras(wt) molecules at the dorsal plasma membrane of a 3T3-A14 cell. The image was taken using 514 nm illumination for 3 mseconds at 2 kW/cm². Scale bar: 1 µm. (B) Example of a single-step photobleaching event of an individual eYFP-H-Ras(wt) molecule. (C) Probability density of the single molecule fluorescence intensity of eYFP-H-Ras(wt). Analysis of the signals of 3028 individual eYFP-H-Ras(wt) molecules at the dorsal plasma membrane of 3T3-A14 cells (solid line) in unstimulated conditions. The probability density of the fluorescence intensity is nearly Gaussian-shaped with a maximum of 204 counts/3 milliseconds. Statistics of the background signal (dashed line) is shown for comparison. The backgound signal had a maximum at 14 counts/3 milliseconds and a width of =10 counts/3 milliseconds. These values translate into a signal to background noise ratio of 20. (D) Trajectory of an eYFP-H-Ras(wt) molecule diffusing in the dorsal plasma membrane of a 3T3-A14 cell. The time between subsequent steps was 10 milliseconds.