Figure 7.

Reproducibility of the fluorescence changes. Four sequential imaging experiments (runs) were done at each NMJ. Within each run, electrical stimuli were applied at a rate of 50 Hz for 2.2 s (stimulus interval, from 2 to 4.2 s). Between the individual runs, the preparation was allowed to recover for 3 min. The calculated fluorescence change is plotted versus time for the following indicators and number of animals, NMJs, and boutons: A, YC2.3, 2, 2, and 15; B, TN-L15, 3, 3, and 27; C, GCaMP1.3, 3, 3, 50; and D, IP, 2, 2, and 23, respectively. The perfect superposition of the individual runs in A and B demonstrates the high reproducibility of the exhibited fluorescence change in the dc indicators YC2.3 and TN-L15. E, The data in A-D are summarized by normalizing the peak amplitudes of each run to the first run. For the measurement of the peak fluorescence, the five nearby frames were averaged. For IP, this peak was subtracted from the signal at the onset of the stimulus. F, Quantification of the raw fluorescence intensity before stimulus onset as a measure of bleaching. The raw fluorescence of each run was normalized to the first run. We did not observe bleaching in any of the GECIs. In all experiments shown, a neutral-density 0.4 filter was used to attenuate the power of the epifluorescence excitation to a level that just allowed us to identify individual NMJs at rest.