Fluorescence Imaging of Calcium Dynamics at Neuromuscular Junctions in the Diaphragm of a Transgenic Mouse

Begin with a fluorescence microscope setup containing an immobilized diaphragm with phrenic nerves from a transgenic mouse. 

The diaphragm's neuromuscular junction consists of a motor neuron, a muscle fiber, and Schwann cells containing acetylcholine receptors and calcium channels.

These cells have fluorescently labeled acetylcholine receptors and express intracellular calcium indicators.

An image splitter attached to the setup enables simultaneous visualization of both fluorescent signals.

The phrenic nerve is positioned in a suction electrode to deliver electrical stimulus.

Under the fluorescence microscope, visualize acetylcholine receptors to identify a neuromuscular junction. 

To set a maximum baseline fluorescence, add a potassium chloride solution to depolarize the cell membrane temporarily. This triggers intracellular calcium influx, causing an increase in the indicators' fluorescence.

Adjust the brightness settings to prevent oversaturation of the fluorescence signal.

After repolarization, apply electrical stimulations to trigger calcium influx into the cell and record the change in intracellular fluorescence at a high frame rate.