Two-Photon Laser Axotomy: A Method to Injure Axons in Zebrafish Embryos and Observe Axonal Recovery
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– Place embryos of interest into phenyl thiourea, PTU, Ringer's solution 到 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 到 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 到 fluoresce red. Increase the intensity of this laser 到 injure the target area without affecting the surrounding tissue. This process is called axotomy. Take a new image with the first laser 到 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 到 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 到 switch from one 到 the other.
Although both lasers are used 到 detect GFP, the two photon emission is visualized with red and the argon laser emission with green in order 到 differentiate the two. Use the argon laser 到 identify an axon 到 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 到 make sure that the axon is still in focus. Click the Stop button so that the Crop tool will be available. Use Crop 到 zoom in on the area of interest. Usually we zoom 到 about 70x. Choose the region of the axon 到 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 到 15% 到 30% transmission. To activate the new settings, click on the Fast XY button and then click Stop quickly afterward 到 avoid excess damage. The axon should be seen as scattered debris if the procedure worked. Finally, 到 ensure that the axon was indeed damaged, switch back 到 the 488 nanometer argon laser. Take another confocal image and create a maximum projection of the Z stacks.
