Imaging Analysis of Insulin Secretion with Two-Photon Microscopy

• Published in: Biological & pharmaceutical bulletin Vol. 38; no. 5; pp. 656 - 662
• Main Author: Takahashi, Noriko
• Format: Journal Article
• Language: English
• Published: Japan The Pharmaceutical Society of Japan 01.05.2015; Japan Science and Technology Agency
• Subjects: Calcium - metabolism; Exocytosis; Glucose - metabolism; imaging; insulin; Insulin - metabolism; Insulin Secretion; Islets of Langerhans; Membrane Fusion; Microscopy, Fluorescence, Multiphoton - methods; secretion; Secretory Vesicles; soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.b14-00880

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 Ca2+ 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.