Tracking Synaptic Vesicles in Live Neurons Using Single-Molecule Super-resolution Microscopy.

Neurotransmitter release relies on the regulated fusion of synaptic vesicles (SVs) that are densely packed within the presynaptic bouton of neurons. The mechanisms by which SVs are clustered at the presynapse while dynamically organizing themselves into different SV pools with distinct fusion probabilities remain unknown. The study of SVs has historically been limited to ultrastructural studies of the presynapse. Examining the nanoscale dynamic organization of SVs in live neurons requires the use of innovative optical labelling approaches, super-resolution microscopy techniques, and appropriate stimulation paradigms that can mimic neuronal physiology. In this chapter, we discuss these aspects by highlighting the use of single-particle tracking photoactivated localization microscopy (sptPALM) to resolve the mobility and clustering of the total pool of SVs, Universal Point Accumulation Imaging in Nanoscale Topography (uPAINT) to study the mobility of SV proteins transiting on the plasma membrane, Dual-pulse subdiffractional Tracking of Internalized Molecules (DsdTIM) to simultaneously track the reserve and recycling pool of SVs and electrical field stimulation for depolarizing primary neurons.