Selective Single-Neuron Laser Ablation in a Zebrafish Larva Using a Confocal Microscope

Take a preheated agarose solution containing an anesthetized transgenic zebrafish larva expressing a fluorescent protein in spinal motor neurons.

Transfer the larva to a glass dish with an agarose ring.

Using a microscope, turn the larva onto its side. Then, allow the agarose to solidify, immobilizing the larva.

Add buffer containing anesthetic to maintain anesthesia.

Place the dish on the confocal microscope stage and use bright-field imaging to locate the dorsal spinal cord region.

Switch to fluorescence mode to visualize fluorescent motor neurons.

Determine the central z-plane of the cell soma for precise ablation targeting.

Configure imaging parameters for high-resolution acquisition while minimizing photodamage.

Once the desired z plane is in focus, perform ablation on the target neuron region using a focused ultraviolet laser.

Laser energy disrupts cellular structures, causing fluorescence loss and cell death. Irreversible fluorescence loss confirms precise ablation without affecting surrounding cells.

Use an adjustable pipette to draw up a larva and let it sink to the bottom of the tip. Drop the larva in a minimal volume of liquid into preheated agarose, then draw up the fish wi