Regulated exocytosis is the process of secretion in specialized cells. Decades of intensive research have defined the molecules that drive the process of membrane fusion, as well as a plethora of accessory factors that shape and modulate the exocytotic function. As regulated exocytosis is generally a rapid event with a millisecond timescale, involving organelles on the nanometer scale, the field has long employed high spatial and temporal resolution techniques, including electrophysiology and, importantly, imaging approaches. Huge gains in the spatial and temporal resolution in biological microscopy have been delivered by recent hardware, engineering, and software breakthroughs; it is now possible to visualize and quantity the distributions, movements, and interactions of large cohorts of single protein molecules inside living cells. These technically demanding approaches have the potential to test long-standing hypotheses in the fields of membrane trafficking and exocytosis, building upon an extraordinary foundation of detailed biochemical and electrophysiological understanding.