Imaging Insulin Granule Dynamics in Human Pancreatic β-Cells Using Total Internal Reflection Fluorescence (TIRF) Microscopy.

Due to the ultra-thin optical sectioning capability of exclusively illuminating space at the interface where total internal reflection occurs, the TIRF microscope has been indispensable for monitoring biological processes adjacent to the plasma membrane with excellent signal-to-noise ratio. Insulin-containing granules fuse with the plasma membrane to release contents within hundreds of milliseconds, which involves well-orchestrated assembly of SNARE complex and associated proteins. A video-rate multiple-color TIRF microscope offers the unique opportunity to visualize single secretory granule docking and fusion dynamics and can also map its regulators with high spatiotemporal resolution. Here, we describe the basic principles and practical implementation of a fast dual-color TIRF microscope, detailing a how-to guide on imaging and analysis of insulin granule dynamics in human β-cells.