Fig 4. Localization of fluorescence transients in low and high [Ca²⁺]_o.

(a) Morphology of individual boutons. Red fluorescence was upsampled (16 x 16 pixels to 128 x 128 pixels), aligned and averaged over all trials. Scale bars represent 0.5 μm. (b) Average response of iGluSnFR superimposed with bouton outline (black line) from red channel (morphology). The bouton outline was generated by thresholding the red channel followed by smoothing. (c) Two-dimensional Gaussian fit to average response. On average, the full width at half maximum (FWHM) was 763 ± 29 nm (n = 12; 5 boutons shown here) (d) Plotting the center position of 2D Gaussian fits to individual trials. Fusion appears to be localized to a small region on the bouton (active zone). Amplitude (ΔF/F₀) of individual trials is color-coded. Scale bars represent 0.5 μm. (e) Increasing the extracellular Ca²⁺ concentration increased the amplitude of individual responses, but did not lead to release events outside the active zone. (f) Fitting responses classified as failures (< 2σ of baseline noise) did not reveal any clustering, indicating that there was indeed no localized signal in these trials (true negatives). (g) Fitting frames before stimulation (green baseline fluorescence) did also not result in clustering.