Total Internal Reflection Fluorescence Microscopy for Visualization of Exocytic Events

Start with a glass-bottom dish containing embryonic cortical neurons on a total internal reflection fluorescence or TIRF microscope stage.

These neurons express a pH-sensitive green fluorescent protein or GFP-tagged vesicle marker that fluoresces in neutral extracellular pH but remains non-fluorescent in acidic vesicles.

Upon vesicle fusion with the plasma membrane, exposure to extracellular pH induces fluorescence, marking the exocytosis event.

Select the objective and set the refractive index to match the neuron.

Direct the laser beam at an angle to the glass, which causes total internal reflection and generates an evanescent wave. This wave selectively excites GFP markers near the plasma membrane.

Set the TIRF penetration depth to exclude GFP markers above the evanescent field.

First, use widefield epifluorescence illumination to locate GFP-expressing neurons.

Then, switch to TIRF illumination for membrane-specific excitation.

Acquire time-lapse images to record the vesicle fusion event and visualize exocytosis.

After setting up the TIRF microscope and the sample and finding a neuronal focal plane according to the text protocol, start the laser software and connect to the laser control