Imaging of Zn^2+ Release from Pancreatic (beta)-Cells at the Level of Single Exocytotic Events

Regulated secretion of Zn^2+ from isolated pancreatic (beta)-cells was imaged using laser-scanning confocal microscopy. In the method, (beta)-cells were incubated in a solution containing the novel fluorescent Zn^2+ indicator FluoZin-3. Zn^2+ released from the cells reacted with the dye to form a fluorescent product, which was detected by the confocal microscope. The new dye is much brighter than Zinquin, previously used for this application, allowing detection limits of 10-40 nM and temporal resolution of 16 ms/image. The high temporal resolution allowed imaging of isolated fluorescent transients that occurred at the edge of the cells following stimulation with 20 mM glucose or 40 mM K+. Fluorescent transients took 16-50 ms to reach a peak from the initial rise and returned to baseline after 170 +- 50 ms (n = 78 transients from 15 cells). It was concluded that the transients correspond to detection of exocytotic release of Zn^2+. Analysis of the temporal and spatial dispersion of the transients indicates that the release of Zn^2+ is not diffusion limited but is instead kinetically controlled in agreement with previous observations of insulin release detected by amperometry.