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. 2008 Jul 16;3(7):e2694. doi: 10.1371/journal.pone.0002694

Figure 2. Monitoring fusion events with a combination of NPY-mRFP and synaptopHluorin.

Figure 2

(a). Fusion of a single vesicle imaged with dual-wavelength image-splitter. synaptopHluorin (SpH) fluorescence appears at t = 1.2 s, and NPY-mRFP fluorescence disappears at the same time. (b). Fluorescence intensity measured at the site of fusion at both wavelengths. (c). Lifetime plot of vesicles that fused in response to stimulation. Data are pooled from n = 10 cells. Each trace starts at the appearance of a vesicle in the TIRF plane (vesicles 1–45 were present at the start of the time-lapse sequence) and ends with the exocytotic event. (d). Normalized time course of secretion, in which fusion events were accumulated and normalized to the number of vesicles visible in each cell before stimulation. Black trace shows the fusion kinetics of all vesicles. Green and magenta traces show the fusion kinetics of resident and newcomer vesicles, respectively. Each trace is fitted with a single exponential (red dotted lines, see Table 2).