Twee-Foton Laser-Geïnduceerde Neurale Verwonding: Een Methode om Axon Degeneratie en Regeneratie in Drosophila Larven te observeren

- Begin by mounting a clean, anesthetized larva under a glass cover slip with its head up. Next, expose the area of interest by gently pushing the cover slip to roll the larva and adjust its position. Use confocal imaging to locate the larva neural cells. Identify a specific axon on one of the target neural cells as the site for injury.

Then expose the target axon to a two-photon laser at a higher power than used for imaging to induce damage. A two-photon laser is used due to its ability to penetrate and precisely localize damage within live tissue with minimal damage to surrounding tissue.

Stop the laser exposure immediately when there is damage to the target neuron, resulting in axotomy, or severing of the axon. Lastly, image the injured neuron to view its degeneration and subsequent regeneration at the desired time points.

In the example protocol, we will mount a larva for microscopy, induce damage to larval sensory neurons, and track neural regeneration.

- Begin with an anesthetizing the larvae. In a fume hood, place a 60-millimeter glass dish into a 15-centimeter plastic petri dish. Then fold a piece of tissue paper and place it in the glass dish. Place the grape agar plate on