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

Anal Chem. 2000 Feb 15;72(4):711-7. doi: 10.1021/ac991085t.

Abstract

Confocal microscopy with Zinquin, a fluorogenic Zn(2+)-specific indicator, was used for spatially and temporally resolved measurement of Zn2+ efflux from single pancreatic beta-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 approximately 0.5 microM. 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 approximately 7 microM. Since insulin is co-stored with Zn2+ in secretory vesicles, it was concluded that the Zn2+ efflux corresponded to exocytosis of insulin/Zn(2+)-containing granules from the beta-cell. Exocytosis sites identified by this technique were frequently localized to one portion of the cell, indicative of active areas of release.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cells, Cultured
  • Exocytosis
  • Extracellular Space / chemistry
  • Fluorescent Dyes*
  • Intracellular Fluid / chemistry
  • Islets of Langerhans / chemistry
  • Islets of Langerhans / metabolism*
  • Mice
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Quinolones*
  • Tosyl Compounds*
  • Zinc / analysis*
  • Zinc / metabolism

Substances

  • Fluorescent Dyes
  • Quinolones
  • Tosyl Compounds
  • Zinc
  • zinquin