One of the current issues in neurobiology is the understanding of regulated exocytosis. The sites of vesicular release are highly specialized molecular complexes (active zones), which are highly regulated and triggered by local intracellular calcium concentration. In order to generate this local and transient increase in calcium concentration, the entry of calcium must co-localize with the active zones, as has been postulated and shown by several investigators. However, an in-vivo demonstration has only been possible recently by using Total Internal ReFlection Microscopy (TIRFM).

We have combined single-vesicle tracking and calcium imaging with variable-angle TIRFM to measure co-localization of sub-membrane calcium entry sites and vesicle release. Variable-angle incidence of the TIRF beam was generated using a bifocal acoustooptical device, obtaining spatial resolutions below 30 nm in the z-axis. Voltametry and capacitance measurements were also performed, and the obtained data renders temporal and spatial parameters showing that:

1. There are two identifiable pools of vesicles, one with higher mobility and farther away from the membrane and the other less mobile and closer to the membrane.

2. Vesicles can transition between these states.

3. Spikes of localized sub-membrane entry generally co-localize with sites at which vesicles are released.