In such cases, however, vesicles would also find protocol move as one object for the entire movie, and thus not contribute any false-positive mobility. Similar to the

use of FIONA in studying the mobility of myosin V (Yildiz et al., 2003), we compiled the locations of each vesicle over the entire movie to form a track of the vesicle’s position over our 20 s of observation time. In order to have sufficient data points to characterize the vesicle’s motion, we discarded any tracks with total length of 12 s or less. Our analysis program also computed the error in the localization of each feature as determined by the system parameters and the feature’s signal-to-noise ratio. Such errors in localization (SD ≈ 20 nm) were small and did not mask the movement of vesicles that were truly mobile (Figure 1C). We note that our approach allowed for nanometer-precision localization and tracking of individual synaptic vesicles in Pexidartinib cell line hippocampal cultures without the need for specialized experimental apparatus. Each experiment

consisted of two sets of movies obtained at 37°C (Figure 1A). First, single-evoked or spontaneous vesicles were sparsely labeled. In both cases, the end of vesicle labeling was marked by the removal of excess dye via a 7 min wash in a low calcium bath solution. We then imaged the stained vesicles at 10 frames/s over a 20 s period (we also performed a series of experiments at twice the frame rate, or twice the duration, both of which yielded identical results; data not shown). In order to differentiate between synaptic vesicles and debris in the culture, every sparse staining experiment was followed by a maximal stain/destain procedure using FM1-43, in which the locations of functional synapses were identified as local maximums that stained/destained Digestive enzyme upon strong stimulation (Figure 1A). Single-particle tracks that colocalized with functional synapses at any time during their lifetime

were taken as true synaptic vesicles (Figures 1B and 1C). A number of vesicles that traveled into and along the axons were observed (Figure S2A) and were excluded from our analysis to avoid the contributions of axonally transported vesicles. We confirmed this assertion by using the microtubule-disrupting agent nocodazole, which was previously shown to block axonal transport (Samson et al., 1979) and had no effect on the mobility of vesicles included for analysis (Figures S2B and S2C). Visual examination of sample tracks from vesicles stained by stimulation or in the presence of TTX indicated that spontaneously labeled vesicles appeared to be less mobile than evoked vesicles. Many evoked vesicles exhibited extensive movements within the field of view (Figure 2A), whereas spontaneous vesicles often appeared stationary (Figure 2B). The observed vesicle motion was not due to the instability of the imaging apparatus, as we confirmed by using tracking of fluorescent 40 nm beads affixed to the coverslip, which exhibited <8 nm/frame drift in each direction (Figure 1C).