Two-Photon Laser Axotomy: A Method to Injure Axons in Zebrafish Embryos and Observe Axonal Recovery

- Place embryos of interest into phenyl thiourea, PTU, Ringer's solution to inhibit pigment formation, which begins at 24 hours post fertilization. This makes the developing anatomy more visible, which is necessary for this procedure. Transfer a tricaine anaesthetized embryo into an imaging chamber. Place a glass slide on top and view it under your microscope. Focus on the axon to be injured, which is visible under a 488 nanometer laser because it transgenetically expresses GFP.

Take a before image of the site. Next, view the axon with a 910 nanometre laser which causes the GFP to fluoresce red. Increase the intensity of this laser to injure the target area without affecting the surrounding tissue. This process is called axotomy. Take a new image with the first laser to confirm the axotomy, which appears as scattered debris. In the following protocol, we will perform an axotomy of peripheral sensory axons in zebrafish embryos using a two-photon laser.

- If a custom built two-photon scope is not available in your laboratory, the Zeiss 510 confocal two-photon microscope can also be used to sever axons. We begin by placing the mounted embryo onto the stage and bringing it into focus using a 25x water objective. Next, turn on the two photon and argon lasers in a multitrack setting so that it is possible to switch from one to the other.

Although both lasers are used to detect GFP, the two photon emission is visualized with red and the argon laser emission with green in order to differentiate the two. Use the argon laser to identify an axon to injure. Under Z settings mark the first and last optical sections, take a confocal image, and create a maximum projection of the Z stack.

Turn off the argon laser and turn on the two photon laser. Scan at an intensity of about 9% transmission to make sure that the axon is still in focus. Click the Stop button so that the Crop tool will be available. Use Crop to zoom in on the area of interest. Usually we zoom to about 70x. Choose the region of the axon to be injured and bring this region into focus. The zoom can be checked under the Mode tab.

Next, under the Channels tab, change the intensity of the two photon from about 9% transmission to 15% to 30% transmission. To activate the new settings, click on the Fast XY button and then click Stop quickly afterward to avoid excess damage. The axon should be seen as scattered debris if the procedure worked. Finally, to ensure that the axon was indeed damaged, switch back to the 488 nanometer argon laser. Take another confocal image and create a maximum projection of the Z stacks.