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