abstract: Exocytosis is a fundamental cell biological process by which cellular vesicles deliver their cargo, e.g., membrane proteins, inflammatory products, or neurotransmitter or hormone molecules, at distinct points in time and space. Total Internal Reflection Fluorescence Microscopy (TIRFM) allows visualizing single exocytotic events simultaneously with local protein densities, Ca2+ levels or plasma membrane composition, giving insight into how proteins and messengers come together to cause the individual events. TIRFM also permits following individual hormone-containing vesicles (“secretory granules”) as they arrive and attach to the cell membrane. We have recently applied mathematical modeling and advanced statistical methods to exploit such rich data. In particular, spatiotemporal simulations of buffered Ca2+ diffusion showed that the risetime rather than the amplitude of fluorescent calcium sensor signals should be used to estimate the distance from calcium channels (CaVs) to granules. From single-granule experimental data, we could then estimate the granule-CaV distance for the each observed granule. Statistical time-to-event analysis (also known as survival analysis), with exocytosis as the event of interest, permitted us to quantify how this distance influences the release probability. Similarly, quantification of the control of exocytosis by local protein densities can be obtained from single-granule data in combination with time-to-event analysis. The methodology can readily be extended to study e.g. granule attachment to the membrane or other distinct, locally controlled biological events. The approach takes advantage of the natural variation between the local environments at the different granules, and shows that biological heterogeneity is a source of information that should not be neglected by averaging, but in contrast can be quantified with appropriate analytical tools.