Single-molecule dynamics of nonglycosylated proteins. Sequential images of a single molecule of an inert mutant of calnexin, calnexin(Y163F), were recorded as in Fig. 4 (the complete sequence can be seen in Video 4). (A) FCS analysis of the calnexin mutant. G(τ) was determined for wild-type calnexin and the calnexin mutant. Wild-type calnexin (black) decayed more slowly than the mutant (blue). (B) Examples of a single image (left), a projection of 1,000 images (right), and a kymograph of the video (bottom). Long-lived spots are indicated by arrows. (C) Translational diffusion of a calnexin mutant. Changes in MSD over time were plotted as in Fig. 4 C. (D) The lifetime of calnexin(Y163F) in the evanescence field was obtained from four streaming images recorded for 15 s. The best-fit d_A was obtained as in Fig. 4 F. (E) Single molecular dynamics of the nonglycosylated cargo YE. Images of YE were recorded as in Fig. 4. Because of its short residence time, the MSD was not traceable for a longer period. (F) Lifetime of YE. Fitting with Eq. 1 failed to predict a reliable value. Error bars represent 95% confidence intervals. Bar, 5 μm.