Fig. 5: Super-resolution localisation of synchronous and asynchronous release events.

a–d Illustration of sub-pixel localisation analysis for single vesicle exocytosis events. a Raw (top) and deconvolved (bottom) SF-iGluSnFR traces from a representative bouton. Only single vesicle release events (black areas on the deconvolved trace) were included in the localisation analysis. These were selected using a 1.25 q amplitude threshold (dashed line), where q (green line) is the quantal size calculated as detailed in Fig. 1e. b Image pre-possessing in time domain. (i) Raw image sequences before and after event 1 (e1, asynchronous release, 3 frames after the preceding action potential, *AP) and event 2 (e2, synchronous release, 1 frame after the preceding action potential). (ii) Corresponding image sequences after pixel-by-pixel deconvolution of the unitary SF-iGluSnFR response. (iii) Event images, obtained by averaging 3 frames centred at the response peak on the deconvolved image stack used for subpixel localisation of release locations using 2D Gaussian fitting (Methods). c Sub-pixel localisation (white crosses) of single quantal events selected in (a) (see also Supplementary Movie 2). Images are after application of a wavelet filter (Methods); e7 could not be localised due to a low signal-to-noise ratio. The bouton outline (white line) was determined by calculating maximal projection of the deconvolved image stack. d Composite image showing relative locations of synchronous (blue) and asynchronous (red) release events within a compact release area. (e) Distribution of release area sizes and their dependency on the overall release efficacy in all boutons that passed the selection criteria for sub-pixel localisation (Methods, Data Inclusion and Exclusion Criteria) (n = 106 boutons from N = 16 cells for wild type and n = 64 boutons from N = 11 cells for Syt1^−/− neurons).