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