To accomplish this, we labeled evoked vesicles by using one action potential (AP) stimulation at the beginning of the 30-s-long dye exposure. Spontaneous labeling was performed via dye exposure for 60 s in the presence of tetrodotoxin (TTX) after 30 s of “presilencing” with TTX to ensure complete activity block (Figure 1A). Given the low release probability of excitatory hippocampal synapses (Murthy et al., 1997) and a very low rate of spontaneous endocytosis at these synapses (∼1 vesicle/min) (Murthy and Stevens, 1999 and Xu et al., 2009), we expected Lapatinib cell line that these protocols would stain at most one vesicle per synapse

in the majority of the synaptic population. To further ensure that only single vesicles were selected for analysis, we used a previously described feature-detection software (Jaqaman et al., 2008) that was capable

of identifying closely positioned particles at subdiffraction distances. Because synaptic vesicles (∼50 nm in diameter) are much smaller than the diffraction-limited resolution of conventional light microscopy, individual vesicles are expected to appear as puncta with a size and shape very similar to the point spread function (PSF) of our imaging system, which was predetermined using stationary fluorescent 40 nm beads (see Figures buy XAV-939 1C and S1A–S1C). The detection software extracts the locations of objects within an image by fitting each detected feature with one or more appropriately positioned Gaussians, each with same width as the PSF. A mixture-model fitting algorithm which weights the penalty from having multiple PSFs against improvement of the fit in the form of an F test (cutoff p < 0.0005) is used to determine the optimal number of PSF features that would best represent each puncta (Jaqaman et al., 2008). Such iterative PSF fitting has been previously shown to achieve ∼100 nm resolution without the use of specialized superresolution imaging equipment (Thomann et al., 2002). In our experiments, more than one particle was also indeed identified in a small number of synapses

(<10%; Figures S1D and S1E). These cases were not analyzed further to avoid ambiguity of intersecting vesicle trajectories. To make sure that only single-vesicle trajectories were being analyzed, we plotted the histograms of integrated fluorescence values at the sites of functional synapses (as determined by our whole synapse stain/destain procedure; Figure 1A) for both spontaneous and evoked vesicle labeling (Figures S1D and S1E). The prediction of the number of vesicles labeled per functional synapse, as given by the fluorescence values histograms, closely agreed with the PSF feature counts from our detection software (Figures S1D and S1E, inset), providing an independent confirmation of the single-vesicle assertion.