Establishing an automated detection and analysis of clustered vesicles. (A) Replica of Fig. 2 A showing a representative of the fields for which clusters were manually or automatically detected and quantified. Scale bar, 5 μm. (B) Manual detection and quantification of six fields of the in vitro vesicle:vesicle tethering assay shown in A. Error bars represent SD. P values were obtained using a two-tailed Student’s t test. *, P < 0.05; **, P < 0.01; *** P < 0.001; ns = no significant difference. (C) Automated detection and quantitation of the same six fields of view quantified in B (see Materials and methods; error bars represent SD, P values obtained and reported as described for B). (D) Zoomed in image of overlayed FITC (Sec4-GFP) and TRITC (FM4-64) channels of vesicle clusters. (E) Automated detection of clusters that are outlined in yellow and filled in in blue. The fluorescence in these clusters is then reported as a percentage of the total fluorescence in the field of view.