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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
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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.
Biological & Pharmaceutical Bulletin
|May 8, 2015
Summary
This study introduces a novel two-photon imaging method to visualize exocytosis in deep tissues. The technique reveals synchronized exocytic events and molecular dynamics in pancreatic islets.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Physiology
Background:
- High-resolution deep tissue imaging is crucial for understanding cellular processes.
- Visualizing exocytosis in living secretory tissues remains challenging.
- Two-photon excitation microscopy offers deep tissue penetration and reduced phototoxicity.
Purpose of the Study:
- To develop and validate a method for detecting and analyzing exocytic events in deep secretory tissues.
- To investigate the spatiotemporal dynamics of exocytosis and fusion pores.
- To correlate exocytic events with molecular dynamics in living tissues.
Main Methods:
- Combined two-photon excitation microscopy with perfusion of a polar fluorescent tracer.
- Established a method to detect exocytic events within secretory tissues.
- Utilized simultaneous multicolor imaging capabilities of two-photon microscopy.
Main Results:
- Successfully detected exocytic events in glucose-stimulated pancreatic islets.
- Observed synchronization between exocytic events and cytosolic Ca(2+) concentrations.
- Demonstrated that full fusion of single secretory granules is the predominant mode of exocytosis, inhibiting compound exocytosis.
- Visualized distributions and conformational changes of fluorescent-labeled molecules alongside exocytic events.
Conclusions:
- The developed method enables high-resolution deep tissue imaging of exocytosis.
- This technique provides insights into the spatiotemporal regulation of exocytosis and fusion pore dynamics.
- It allows for the analysis of molecular mechanisms underlying membrane fusion during exocytosis in living tissues.
