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

1: Two-photon axotomy using a custom built two-photon microscope with a Chameleon Ti-Sapphire laser

1. Prepare for imaging. Place the mounted embryo(s) on a slide holder under the microscope. Focus on one embryo with a 40X (0.8 NA) water objective. Turn on the laser. We have been able to reproducibly visualize and damage GFP expressing neurons using the following parameters. To visualize axons at a non-damaging power, set laser to a wavelength of 910 nanometers (nm) and a power of 30 milliwatts (mW listed are the amount of power at the sample). Open the imaging software. We use ScanImage Software developed in Karel Svoboda's laboratory.
2. Press focus to scan the embryo with the two-photon laser, locate the axon you wish to axotomize, and capture an image of this axon. Mark the first and last Z positions, acquire the image, and make a maximum projection of the Z stacks.
3. Zoom in 70X on the branch of the axon you wish to axotomize and stop scanning. Increase the mW at the sample to the damaging power of 180 mW, and perform a single scan with the laser. We do this by setting the number of Z slices to 1 and pressing “Grab”. This should be sufficient to sever the axon. See discussion for methods to optimize this procedure for your microscope and experimental goal.
4. Zoom out, reduce the power to 30 mW and take an image.

2: Two-photon axotomy on Zeiss 510 confocal/two-photon microscope

1. Place mounted embryo onto the stage and bring it into focus using a 25X water objective or other suitable objective.
2. Turn on the two-photon (910 nm) and Argon (488 nm) lasers in a multi-track 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 to differentiate the two.
3. 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.
4. Choose the region of the axon to be injured and bring this region into focus. Turn off the Argon laser and turn on the two-photon laser. Scan the specimen with the two-photon at a laser intensity of ~9% transmission to make sure that the axon is still in focus.
5. 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 ~70X (zoom can be checked under the “mode” tab). Under the “channels” tab change the intensity of the two-photon from ~9% transmission to 15-20% transmission.
6. To sever an axon activate the “fast XY” button for about 1 second and then press the stop button to avoid excess damage. The axon should be seen as scattered debris if the procedure worked.
7. To ensure that the axon was damaged switch back to the 488 nm Argon laser, take another confocal image, and create a maximum projection.