Total Internal Reflection Fluorescence Microscopy for Visualization of Exocytic Events

Abstract

Source: Winkle, C. C., et al. Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis. J. Vis. Exp. (2016)

This video demonstrates total internal reflection fluorescence (TIRF) microscopy for visualizing exocytic events in cortical neurons. A pH-sensitive green fluorescent protein (GFP) marker fluoresces upon vesicle fusion, with selective TIRF excitation capturing membrane-proximal exocytosis in time-lapse imaging.

Protocol

1. Imaging Exocytic Events via Total Internal Reflection Fluorescence (TIRF) Microscopy

Note: This protocol requires specialized microscopy equipment, including an environmental chamber to maintain temperature, humidity, and carbon dioxide (CO₂), an inverted total internal reflection fluorescence (TIRF) microscope equipped with an epifluorescent illumination, a high magnification/ high numerical aperture (NA) TIRF objective, an automated XYZ stage, and a sensitive charge-coupled device (CCD) detector. This protocol uses a fully automated inverted microscope equipped with a 100x 1.49 NA TIRF objective, a solid state 491 nm laser, and an el....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35mm glass bottom live cell imaging dishes	Matek Corporation	p356-1.5-14-C	Must be coated with 1mg/mL poly-D-lysine and rinsed prior to plating cells
Olympus IX81-ZDC2 inverted microscope	Olympus
Lambda LS xenon lamp	Sutter Instruments Company
Environmental stage top incubator	Tokai Hit
100x 1.49 NA TIRF objective	Olympus
Andor iXon EM-CCD	Andor
Cell TIRF control software	Olympus		Software used to control lasers for TIRF imaging
Fiji (Image J)	NIH	ImageJ version 1.49t
Rabbit polyclonal anti-human VAMP2	Cell signaling	11829
Poly-D-lysine	Sigma	p-7886	Dissolved in sterile water at 1mg/mL