This article describes a method for inducing localized neuronal injury in Drosophila larvae using two-photon microscopy. The procedure involves anesthetizing the larvae, positioning them on a slide, and using a laser to target specific axons, allowing for the study of neuronal responses to injury.
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.
Begin with 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 the tissue after the diethyl ether is added. Next, onto a glass slide, place one drop of halocarbon 27 oil at the center, and place a spot of vacuum grease on each of the four corners.
Then, use forceps to transfer one larva onto the agar plate, and cover the glass dish to anesthetize the larva. As soon as the larva stops moving, carefully transfer it to the halocarbon oil with its head upright. Then, place a coverslip over the slide, and press it down gently, until it touches the larva. Next, use gentle force to slide the coverslip to roll the cells to be ablated to where the two photon laser will most easily hit them. The location will vary depending on what neurons are being targeted.
Now, secure the assembly on the two photon microscope stage, and focus on the cells of interest using a 40x oil immersion objective. In the software, switch to the scanning mode, and load the saved protocol. Make sure the pinhole is opened all the way. Then, in Live mode, get a good image of the region of interest. Next, stop the Live scan, so that the Crop button will become available. Using the Crop function, adjust the scan window to focus the target area on just the prospective site of injury.
Then open a new imaging window. Now, reduce the scan speed, and increase the laser intensity. Then toggle the continuous button to start and stop the scan. Watch carefully. As soon as there is a drastic increase in fluorescence, end the scan. Next, switch back to the original imaging window, and select the Live mode, and find the region that was just targeted by adjusting the focus.
A good indication of successful injury is the appearance of a small crater, ring-like structure, or localized debris right on the injury site. If the laser power was too high, a large damage area would be visible, which can be lethal.
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