Analysis of the motion of a single YE(gly)₂ molecule. Images of YE(gly)₂ in the evanescent field were recorded at 32°C and a resolution of 15 ms (Videos 1 and 2). (A) Four sequential images from Video 1. A projection of 1,000 images reveals traces of a reticular network (bottom). (B) The displacement of each spot in sequential images (Video 2). An image (green) was compared with the next consecutive image (red). (C) Translational diffusion of YE(gly)₂. Averaged MSD was plotted against time intervals (T). Error bars represent 95% confidence intervals. (D) Appearance and disappearance of single molecules (numbered arrows in A) over nine frames. (E) A one-dimensional image of a single pixel width of the video was aligned with the progression of time. The kymograph is an image of 7 s in length and 19 μm in distance. (F) Lifetime distribution of each spot. Lifetime of the fluorescence spots was determined from four streaming images, each of which was obtained over 15 s, and the time distribution was counted. The lifetime dataset was normalized, and the best-fit value of d_A was calculated. Bar, 5 μm.