Fig. 2
Multi-synapse imaging of quantal glutamate release with SF-iGluSnFR.A184S. a CA3 pyramidal cell axon fragment in area CA1 showing four presynaptic boutons (b1–b4); the scanning dwell points in the bouton centres (red dots, dwell-delay time ~1.5 ms per bouton) and laser scan trajectory (dotted yellow line) illustrated; scale bar, 5 µm. b A pseudo-linescan image of SF-iGluSnFR.A184S signals recorded simultaneously (one sweep example) at four boutons shown in a as indicated, during somatic generation of two action potentials 50 ms apart (top trace, current clamp). Arrow diagram relates displayed pixels to the scanning cycle: pixels at each displayed ith line are recorded sequentially among boutons (small arrows), and the next cycle fills the (i+1)th line of the display. Thus a pseudo-linescan image is generated showing brightness dynamics at individual boutons, with ~1.5 ms resolution; glutamate releases and failures can be seen; scale bars, 60 mV (vertical) and 100 ms (horizontal). c A summary of 22 trials in the experiment shown in a, b; green traces, single-sweep SF-iGluSnFR.A184S intensity readout at the four bouton centres; black traces, all-sweep-average; P1:P2, average probability Pr (release success rate) of the first (red) and second (blue) release events; scale bars, 50% ΔF/F (vertical) and 100 ms (horizontal). d Amplitude histograms (SF-iGluSnFR.A184S ΔF/F signal, first and second response counts combined; prepulse baseline subtracted) with a semi-unconstrained multi-Gaussian fit (blue line, Methods) indicating peaks that correspond to estimated quantal amplitudes; the leftmost peak corresponds to zero signal (failure; yellow shade); dotted lines, individual Gaussians; arrows, average amplitudes (including failures) of the first (red) and second (blue) glutamate responses
