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Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets
Published on: September 13, 2017
Imaging analysis of insulin secretion with two-photon microscopy
1Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo.
High-resolution deep tissue imaging is possible with two-photon excitation microscopy. With the combined application of two-photon imaging and perfusion with a polar fluorescent tracer, we have established a method to detect exocytic events inside secretory tissues. This method displays the spatiotemporal distribution of exocytic sites, dynamics of fusion pores, and modes of exocytosis. In glucose-stimulated pancreatic islets, exocytic events were observed to be synchronized with an increase in cytosolic Ca(2+) concentrations. Full fusion of a single secretory granule is the typical mode of exocytosis and compound exocytosis is inhibited. Because two-photon excitation enables simultaneous multicolor imaging due to the broadened excitation spectra, the distributions and conformational changes in fluorescent-labeled molecules can be simultaneously visualized with exocytic events. Therefore, we can analyze the dynamics of the molecules involved in membrane fusion and their association with exocytosis in living tissues.
High-resolution deep tissue imaging is possible with two-photon excitation microscopy. With the combined application of two-photon imaging and perfusion with a polar fluorescent tracer, we have established a method to detect exocytic events inside secretory tissues. This method displays the spatiotemporal distribution of exocytic sites, dynamics of fusion pores, and modes of exocytosis. In glucose-stimulated pancreatic islets, exocytic events were observed to be synchronized with an increase in cytosolic Ca(2+) concentrations. Full fusion of a single secretory granule is the typical mode of exocytosis and compound exocytosis is inhibited. Because two-photon excitation enables simultaneous multicolor imaging due to the broadened excitation spectra, the distributions and conformational changes in fluorescent-labeled molecules can be simultaneously visualized with exocytic events. Therefore, we can analyze the dynamics of the molecules involved in membrane fusion and their association with exocytosis in living tissues.
