(A) Neurons stimulated (10 Hz, 1 s) with 400 nM Qdots present, then thoroughly washed. The distribution of photoluminescent intensity was measured at FM4-64-defined ROIs. Best fit obtained with three evenly spaced Gaussians (0, 1 and 2), 63.9 a.u. apart. Spacing agrees with amplitude of blinking events (grey bars; mean, 61.3±4.5 a.u)(p>0.25, t-test). Inset, circles mark functional boutons identified by FM4-64 staining, with (red) or without (white) single Qdot-loading. Scale bar=3 μm. (B) pH-dependent Qdot photoluminescence. Cartoons: hypothetical Qdot signals arising from pH-dependence. (C) Photoluminescence traces recorded during 0.1-Hz, 2-min field stimulation (dashed lines). (D) Upon stimulation, changes in Qdot signal (ΔF) could be classified as noise (gray bars, centered at 0 a.u.) or a clear positive deflection (red bars, centered at ~9 a.u., >2.5 s.d. of noise), ~15% of size of subsequent negative deflections (blue bars, centered at ~-63 a.u.).

(A) Raster representation of traces (n=302) from single Qdot loaded vesicles that responded to 0.1-Hz 2-min field stimulation. For each stimulus and subsequent interval, Qdot signals registered as non-response (gray), K&R (red), non-response following K&R (maroon), FCF (blue), or Qdot no longer present in ROI (black). Pooled traces from N=8 coverslips, 3 separate cultures. (B) Traces corresponding to the first 12 rasters in A. Photoluminescence changes color-coded for each stimulus and ensuing interval as in A. (C) Numbers of K&R (red square) and FCF events (blue triangle), plotted for every stimulus. (D) K&R ratio for every stimulus (N=8). Vertical bars, s.e.m. (E) Numbers of K&R (red squares) and FCF events (blue triangles), plotted for pre-stimulation hypertonic challenge (suc) and for each field stimulus. (F) Corresponding K&R ratio (filled red squares), compared with control (faded red squares, copied from C2)(**, p<0.01, χ2-test).

(A) Insets, samples taken in normal Tyrode with single shocks, inter-stimulus interval >20 s. Two types of fits were overlaid: a single exponential decay (gray), and a plateau followed by an exponential (black), the latter fitting significantly better even after statistical penalization for the extra parameter (AIC score, -60.5; p<0.001). Comparison of pooled data (black symbols, n=43), and averages of the two kinds of fits (gray and black). (B) Samples in 50 mM Tris (green) and in 10 mM HEPES (black). (C) Corresponding pooled data in Tris (n=37) and in HEPES (same as A). (D) Samples (normal Tyrode) with 10-Hz (pink) and single-pulse stimulation (black). Smooth lines, corresponding fits as above. (E) Pooled data taken with 10 Hz (n=46) and single pulse stimulation (same as A). (F) Cumulative distributions of fit parameters. Plateau amplitude: distributions were not different (all p>0.1, K-S test); plateau duration: only distribution for 10-Hz stimulation was longer (p<0.01, K-S test); Decay τ: only distribution for 50 mM Tris was slower (p<0.01, K-S test).

Cartoons: hypothesized effect of acute or chronic block of the vesicular H⁺-ATPase with bafilomycin A1. Without Baf (normal), Qdot photoluminescence is diminished (maroon) by acidic luminal pH (gray). Deacidification upon vesicle fusion removes this quenching and Qdot brightens (red). Acute application of Baf (acutely blocked) prevents reacidification after vesicle retrieval; chronic Baf (chronically blocked) removes all pH gradients. Experimental traces illustrate typical photoluminescence patterns under three conditions. Patterns classified as K&R, FCF, or K&R+FCF (labeled rows) based on analysis of collected data (Fig S4).

(A) Sample traces from Qdot-loaded vesicles. (B) Categorization of vesicles with different fusion behaviors. (C) The K&R ratio during 10-Hz 2-min stimulation (analyzed with 5 s time bins to improve S/N) was significantly higher than that during 0.1-Hz stimulation (faded symbols, data from Fig 4C2). (D) Latency of first fusion in the different categories (same color coding as B). Each plot normalized by the number of vesicles in that category. The higher its P_r/v, the more K&R events a vesicle could support (P_r/v=0.051, 0.023, 0.010 and 0.001 for 3K+F, 2K+F, K+F and FCF only, respectively). (E) the interval between 2 consecutive K&R events (K-K, red) was significantly shorter than that between K&R and a subsequent FCF of the same vesicle (K-F, blue)(p<0.01, K-S test).