Detection of secretion from single pancreatic β-cells using extracellular fluorogenic reactions and confocal fluorescence microscopy

Confocal microscopy with Zinquin, a fluorogenic Zn²⁺-specific indicator, was used for spatially and temporally resolved measurement of Zn²⁺ efflux from single pancreatic β-cells. When cells were incubated in buffer containing Zinquin, application of insulin secretagogues evoked an increase in fluorescence around the surface of the cell, indicative of detection of Zn²⁺ efflux from the cell. The fluorescence increases corresponded spatially and temporally with measurements of exocytosis obtained simultaneously by amperometry. When images were taken at 266-ms intervals, the detection limit for Zn²⁺ was ~0.5 μM. With this image frequency, it was possible to observe bursts of fluorescence which were interpreted as fluctuations of Zn²⁺ level due to exocytosis. The average intensity of these fluorescence bursts corresponded to a Zn²⁺ concentration of ~7 μM. Since insulin is co-stored with Zn²⁺ in secretory vesicles, it was concluded that the Zn²⁺ efflux corresponded to exocytosis of insulin/Zn²⁺-containing granules from the β-cell. Exocytosis sites identified by this technique were frequently localized to one portion of the cell, indicative of active areas of release.