Fig. 3

Generation and analysis of line scan data. (A) Scan lines were positioned perpendicular to the plasma membrane, extending from the intracellular side of the ribbon into the extracellular space. For illustrative purposes, a second scan line is shown at a non-ribbon location. Lower-case letters show line positions for panels B-D. (B) Intensity profile of green RBP fluorescence at the non-ribbon location. The parameter x_1/2 from the sigmoid fit (black line) is taken as the position of the membrane (see Materials and methods). (C) Intensity profile of green RBP fluorescence at the ribbon. The parameter x₀ is the peak of the Gaussian fit, giving the x-position of the center of the ribbon (see Materials and methods). (D) Intensity profile of paRFP fluorescence at the ribbon, with the x-position of the labeled vesicle given by x₀. (E) Example of x-t image of paRFP spot at a ribbon, consisting of 64 line scans (right) taken over 472 ms. X-axis intensity profiles are plotted (left) for the first 16 line scans to illustrate the noise within and across individual line scans. The superimposed black line shows the average of all 64 lines. (F) Data from E were averaged over four lines to reduce noise and allow more precise localization of the paRFP spot along the x-axis. The intensity profiles show the first four of the temporally averaged line scans, with the average for all lines superimposed in black. (G) Example x-t images averaged over five line scans, showing a stable paRFP-labeled synaptic vesicle associated with a ribbon, which was labeled in green with RBP. Membrane potential was voltage-clamped at -60 mV. (H) Intensity profiles of RBP (green) and paRFP (red) taken in successive 406-ms intervals from the images in G. The red Xs show the position of x₀ for each paRFP trace, which varied over a range of 48 nm during the 15-s recording.