Single synaptic vesicle tracking in TIRFM-amenable hippocampal synapses.

Abstract

Single synaptic vesicle (SV) tracking is used to explore the dynamics of SV movement close to active zones (AZ) by total internal reflection fluorescence microscopy (TIRFM). To do so, we used cultured hippocampal ‘xenapses’, purely presynaptic boutons formed by neurons on micro-patterned coverslips, functionalized with synaptogenic cell adhesion proteins. For single SV labeling we developed surface-modified single-color quantum dots with improved photostability and pH-sensitivity for single SV tracking. Obtained single SV movies are analyzed with an improved single-particle tracking software, which combines Automatic Correction for sCMOS-related Noise algorithm (Nat. Commun., Mandracchia et al., 2020) for denoising, U-Track (Nat. Methods, Jaqaman et al., 2008) for frame-to-frame particle linking, and a newly developed single-particle localization algorithm. We yield a localization accuracy of 0.1229 ± 0.1004 pixels (110 nm pixel size) and are able to resolve merging and splitting events of two crossing particle trajectories even though they have low signal-to-noise ratio of ∼1.2. In hippocampal xenapses with active zones (AZ) facing the coverslip, SVs appear to jump between discrete levels of axial distance to AZs, and these discrete levels are remarkably stable over tens of seconds. As SVs in electron micrographs appear to be interconnected by short tethers, these discrete levels might arise from a layered structure of the SV cluster above the AZ.