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. 2016 Feb 11;5:e13245. doi: 10.7554/eLife.13245

Figure 5. Synaptic vesicle exocytosis reported by pHluorin occurs at a distance from the plasma membrane.

(A) Example of x-t images showing a pHluorin event from SypHy (green) at a ribbon shortly after onset of depolarization from -60 mV to -15 mV, shown by the trace to the right. Ribbon fluorescence from deep-red CF633-RBP is pseudocolored red. (B) Example of x-t images showing a pHluorin event from SypHy (green) on the membrane-distal side of a ribbon (red) in a different cell during depolarization to -15 mV. (C) (D) Fluorescence intensity profiles along the x-axis for RBP (red) and pHluorin (green) for the examples shown in A and B. Black lines are fits as described in Figure 3A–D and in Materials and methods. The x-axis position of the pHluorin event was taken to be the peak of the Gaussian from the fit (x0, shown by the dotted green line), and the position of the plasma membrane was taken to be the parameter x1/2 (dotted black line) from the fit to the fluorescence profile of the ribbon (red traces). (E) Histogram of the x-axis position of 127 pHluorin events from SypHy and Vglut1-pHluorin during sustained depolarization. The solid arrow shows the average relative position of the plasma membrane, estimated as described in Materials and methods (error bar: ± 1 sem). The dashed arrow shows the estimated position of the membrane at the top and bottom of the optical section, arrived at by assuming that membrane curvature observed along the y-axis for each x-y image also applied in the z-axis. Most of the pHluorin events fell outside the range of membrane positions between the two arrows.

DOI: http://dx.doi.org/10.7554/eLife.13245.012

Figure 5.

Figure 5—figure supplement 1. Noise level during pHluorin events.

Figure 5—figure supplement 1.

Green traces show x-axis intensity profiles during the example pHluorin events shown in Figure 5. (A, B) Event from Figure 5A. (C,D) Event from Figure 5B. (A, C) The fluorescence profile was averaged over successive groups of four scan lines during the pHluorin event. The black line shows the average over the entire duration of the event (A: 48 scan lines; C: 128 scan lines). The noise level after averaging over four scan lines was insufficient to allow precise localization of the pHluorin emitter. (B, D) The same data from A and C averaged over successive groups of 16 scan lines (B) or 32 scan lines (D), which reduced noise and allowed more precise localization of the event along the x-axis.
DOI: http://dx.doi.org/10.7554/eLife.13245.013
Figure 5—figure supplement 2. Variation in ribbon position in different focal planes.

Figure 5—figure supplement 2.

(A) X-axis profiles of RBP fluorescence were measured after displacing the objective in the z-axis by the indicated relative amounts, spanning the full extent of the labeled ribbon in the z-axis. The gray Xs show fitted values of x0 for the six traces (positions 200–1200 nm) when the ribbon was visible. This is an example of a ribbon that showed little variation in x0 along the z-axis. (B) X-axis profiles of RBP fluorescence obtained in the manner described for A, but for a ribbon that showed larger variation in x0 along the z-axis. (C) Summary of the variation in x0 of the ribbon along the z-axis for 13 experiments. Data were normalized for each experiment by measuring the change in x0 (Δx0) relative to x0 at the brightest focal position for that experiment.
DOI: http://dx.doi.org/10.7554/eLife.13245.014