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 Confocal Microscope, or LSCM software, choose electrophysiology and acquisition mode to set imaging parameters. In the Job menu settings, select the Trigger settings to trigger the stimulator with the microscope sync pulse and set the Trigger Out on Frame Field to the OUT 1 channel.

Turn the scanning mode to xyt at a frequency of scanning of 1,400 Hertz. The zoom factor should be 6.1 with the pinhole fully open. Ensure that sequential transpassing bidirectional x mode is on. Then set the minimum time to form a frame at 52 milliseconds and collect frames in a raw video at 20 frames. Set excitation wavelength of the argon laser at 488 nanometers, with 8% of output power.

When the parameters are set, press the Live Mode button to get a live preview of nerve terminals loaded with the dye. In the live mode, search for the Region Of Interest, or ROI, to obtain the best focus, then shift the delay on the stimulator by 2 milliseconds less relative to the previous value. And run the data acquisition software to acquire 26 sequences by shifting each sequence two milliseconds from the previous one.