Imaging Fast Calcium Transients in Mouse Nerve Endings Using Confocal Microscopy

Begin with a silicone elastomer-coated experimental chamber containing a secured mouse muscle positioned under a confocal microscope.

The muscle is immersed in a physiological solution containing inhibitors that block muscle contractions.

The nerve stump labeled with a fluorescent calcium dye is positioned inside the suction electrode, which is connected to an electric stimulator.

At the neuromuscular junction, the peripheral nerve is connected to the muscle.

Apply an electrical stimulus to the nerve using the electrode. This triggers a rapid influx of calcium ions that bind to intracellular calcium dye.

Illuminate the neuromuscular junction to excite the calcium dye, causing it to fluoresce.

Then, capture high-resolution images to record rapid calcium transients in the peripheral nerve endings.

Select the region of interest and measure the fluorescence intensity.

A rapid increase in fluorescence intensity indicates calcium influx at the peripheral nerve endings, while a decrease reflects calcium clearance.

Fill up the perfusion system with the Ringer's solution containing 10 micromolar D-tubocurarine and switch on the perfusion suction pump to start perfusion. In the Laser Scanning Confoc