Two-Photon Laser-Induced Neural Injury: A Method to Observe Axon Degeneration and Regeneration in Drosophila Larvae
筆記録
– 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 に roll the larva and adjust its position. Use confocal imaging に 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 に a two-photon laser at a higher power than used for imaging に induce damage. A two-photon laser is used due に its ability に penetrate and precisely localize damage within live tissue with minimal damage に surrounding tissue.
Stop the laser exposure immediately when there is damage に the target neuron, resulting で axotomy, or severing of the axon. Lastly, image the injured neuron に view its degeneration and subsequent regeneration at the desired time points.
In the example protocol, we will mount a larva for microscopy, induce damage に 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 で 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 に transfer one larva onto the agar plate and cover the glass dish に anesthetize the larva. As soon as the lava stops moving, carefully transfer it に the halocarbon oil with its head upright. Then place a cover slip over the slide and press it down gently until it touches the larva.
Next, use gentle force に slide the cover slip に roll the cells に be ablated に 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 に the scanning mode and load the saved protocol. Make sure the pinhole is opened all the way. Then, で 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 に 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 に start and stop the scan. Watch carefully. As soon as there is a drastic increase で fluorescence, end the scan.
Next, switch back に 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 damaged area would be visible, which can be lethal.
– Now carefully remove the cover slip, and transfer the injured larva onto a new plate with yeast paste. Put the plate into a 60 millimeter dish along with a propionic acid-soaked tissue. Then return the plate に culturing temperature.
For subsequent imaging of the larva, make use of the saved confocal setup and collect z stack images with a 25x objective. Be sure に include the normalization point so the regeneration can be quantified.
