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Place an anesthetized Drosophila larva with its head-up on an oil drop on a glass slide containing vacuum grease spots.
Position a coverslip and gently press it to make contact with the larva.
Adjust the larva to expose the region containing the target neurons expressing fluorescent proteins.
Secure the slide on a two-photon microscope.
Use a low-power laser to minimize damage and scan the larva to locate the region with the fluorescent target neurons.
Next, adjust the scan window to focus on the target region containing axons, a type of neuronal projections.
Now, reduce the scan rate and increase the laser power.
The high-power laser will strike the targeted axons, generating localized heat that damages them and enhances fluorescence around the injury site.
Finally, switch back to low-power laser mode. The presence of small crater, ring-like structures at the injury site confirms successful neuronal injury.
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