Figure 1. Tracking and mobility analysis of vesicles in a representative cell.

(a). First image in a time-lapse image sequence of a representative control cell expressing NPY-Venus. Vesicles (circled) were identified by their fluorescence profile and tracked throughout the sequence. Vesicles that appeared later in the sequence were also identified and tracked. (b). A kymograph representing the duration of vesicle residence near the membrane. Individual vesicles are represented as lines initiating at vesicle appearance and terminating at vesicle disappearance from the TIRF plane (vesicles 1–39 were present at the start of the time-lapse sequence). (c). Histogram depicting the lifetime distribution of vesicles in the representative cell shown in a. (d). Average logarithmically binned histogram showing the distribution of vesicle diffusion coefficients in control cells (n = 18 cells). (e). Time-coded trajectory for the vesicle marked with a green circle in a. The vesicle was tracked for 60 s (see f for color code). Black circle indicates the size of an average chromaffin vesicle. (f). For the same vesicle shown in e, X and Y coordinates are shown separately (middle and bottom traces, respectively) with a windowed-velocity graph (top) that is color-coded as in e. Asterisks denote periods of high mobility. (g). Normalized cumulative-velocity histograms of all single vesicles in one cell (gray curves) and the mean histogram for the same cell (black; error bars represent SEM). Blue and red histograms describe the mobility of two representative vesicles with high and low mobility, respectively.