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 software. Set the illumination to wide field, and select the objective. Then set the refractive index of the sample, and adjust the laser intensity by unchecking TTL for the 491 laser.

Imaging parameter optimization is important during imaging of floor intact exocytic vesicles to avoid focus drift, and phototoxicity while producing images of high signal-to-noise ratio sufficient for analysis.

Adjust the slider to 100, and then bring it back down to a value between 20 and 40. Then recheck TTL. Next, focus on the sample again in transmitted light illumination.

Then in the imaging software, select the 491 laser illumination, and open the shutter. Place the condenser upside down on the optical bench so as not to scratch the lens. Fine adjust the focal point of the laser on the ceiling.

And with the condenser removed, center the point to the center of the closed field diaphragm. Then replace the condenser. And in the TIRF software, set the penetration depth to 110 nanometers. Then switch from wide-field illumination to TIRF illumination mode for imaging.

Now, using wide-field epifluorescence, find VAMP2 florin-expressing cells through the oculars. Then to reduce photobleaching and phototoxicity, adjust the imaging parameters to maximize the signal-to-noise ratio and dynamic range using minimal exposure time and laser intensity. Set continuous autofocus per cell. Then acquire a time lapse image set with acquisition occurring every 0.5 seconds for 5 minutes.