Two-Photon Laser Axotomy: A Method to Injure Axons in Zebrafish Embryos and Observe Axonal Recovery
Trascrizione
– Place embryos of interest into phenyl thiourea, PTU, Ringer's solution a 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 a 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 a fluoresce red. Increase the intensity of this laser a injure the target area without affecting the surrounding tissue. This process is called axotomy. Take a new image with the first laser a confirm the axotomy, which appears as scattered debris. In the following protocol, we will perform an axotomy of peripheral sensory axons di zebrafish embryos using a two-photon laser.
– If a custom built two-photon scope is not available di your laboratory, the Zeiss 510 confocal two-photon microscope can also be used a 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 di a multitrack setting so that it is possible a switch from one a the other.
Although both lasers are used a detect GFP, the two photon emission is visualized with red and the argon laser emission with green di order a differentiate the two. Use the argon laser a identify an axon a 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 a make sure that the axon is still di focus. Click the Stop button so that the Crop tool will be available. Use Crop a zoom di on the area of interest. Usually we zoom a about 70x. Choose the region of the axon a 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 a 15% a 30% transmission. To activate the new settings, click on the Fast XY button and then click Stop quickly afterward a avoid excess damage. The axon should be seen as scattered debris if the procedure worked. Finally, a ensure that the axon was indeed damaged, switch back a the 488 nanometer argon laser. Take another confocal image and create a maximum projection of the Z stacks.
