High-speed, simultaneous, two-color, single-molecule imaging showed transient recruitment of Lyn-FG in/on the inner leaflet at CD59 clusters located in/on the outer leaflet. (A) Typical single-molecule image sequences (6.45-ms resolution; every other image is shown) showing the colocalization of a CD59 cluster (top row and magenta spots in the bottom row) and a single molecule of Lyn-FG (green arrowheads in the middle row and green spots in the bottom row). Lyn-FG spots appear brighter during colocalization due to slower diffusion. (B) The trajectories of the CD59 cluster (magenta) and the Lyn-FG molecule (green) shown in A. These molecules became colocalized (orange circular region with a radius of 150 nm around the CD59 cluster position) between 39 and 91 ms (52 ms; orange box). (C) Another display of the colocalization event shown in A and B, showing the displacements of an Lyn-FG molecule and a CD59 cluster along the x and y axes (left and right, respectively) from the average position of the CD59 cluster during the colocalization period, plotted against time. Circles indicate the times employed in B.