Fig. 3.

Method for evaluating the presence or absence of bridges between vesicles. a Screenshot of graphical user interface for evaluating presence or absence of bridges. Mini-tomograms for each vesicle pair were rotated so that the centers of both vesicles were within the horizontal plane. Images of vesicle pairs were then presented in a random order simultaneously from three angles: not rotated; and rotated $\pm {45}^{\circ }$ about the central axis common to both vesicles. When the “3-D display” radio button is selected, the scroll bar to the right allows the user to scroll through the individual virtual sections. Otherwise, the mean images of all virtual sections between 0.25 median vesicle diameters above and below the central plane are displayed. The lower scroll bars below the images control contrast. b–e Initial survey of 13 WT and 15 DKO tomograms for vesicle pairs separated up to 1.5 vesicle diameters. bZ-angle for a pair of vesicles is defined as the angle between the axis passing through the centers of both vesicles and the plane that is horizontal to the tissue slice. c Fraction of WT vesicle pairs for which images were evaluated as qualitatively “Bad” or “Terrible” vs Z-angle. d Probability of detecting a bridge vs Z-angle in WT synapses. Scores of 1 (“Certain”) and 2 (“Likely”) were counted as bridges. e Probability of detecting a bridge vs distance between the vesicles for Z-angles $\le {25}^{\circ }$ (640 pairs for WT, 374 for DKO). Distance units are median vesicle diameters, which were calculated across all vesicles for each tomogram independently