Targeted Neuronal Ablation Using Two-Photon Microscopy in a Zebrafish Larava

Begin with an anesthetized transgenic zebrafish larva immobilized in agarose and placed in a Petri dish containing an anesthetic solution.

Position it under a two-photon laser microscope, which uses two photons for detailed imaging.

Focus on the top and bottom layers of the brain to set imaging boundaries.

Capture images at various depths to observe neurons before laser treatment.

Adjust the focus on the region of interest and adjust the frame size to refine the focus on neurons.

Select and irradiate the neurons using the two-photon laser.

The Laser induces localized heat, resulting in neuronal death with minimal damage to surrounding tissue. Capture the image.

Compare pre and post-treatment images to assess fluorescence changes.

Refocus neurons in deeper planes and repeat the irradiation to treat all neurons in the region of interest..

Recapture the image and compare it with the pre-treatment image. The loss of fluorescence after laser treatment confirms successful neuronal ablation.

Place the agarose-embedded larva under a 2-photon laser-scanning microscope and start the microscopy system. Open the image acquisition software, and under the Laser tab, click the on button to turn on the laser. Wait for the status of the laser to change from Busy to Mode Locked.

On the Channels tab, set the laser's wavelength to 880 nanometers for EGFP ablation, or 800 nanometers for GcAMP ablation. Select the 20x objective lens by manually moving the lens revolver. Then, click the Locate tab. Click GFP to change the optical pathways, and directly view the fluorescence by eye. Center the zebrafish larval brain in the field of view. Then, click the Acquisition tab to go back to two-photon microscopy and set the laser power and gain.

As a record of the before ablation condition, click Z-Stack to select the Z-Stack option. Then, after focusing on the ventral end of the larval brain, click the Set First button to set the lower limit. And after focusing on the dorsal most surface of the brain, click the Set Last button to set the upper limit. Finally, click the Start experiment button to run the Z-Stack image acquisition.

Next, select the 63x objective lens by manually moving the lens revolver. Then, click the Locate tab to switch to epifluorescence microscopy. Center the cells to be ablated by eye. Then, click the Acquisition tab to go back to laser scanning microscopy.

In the Acquisition Mode tab, set the frame size to 256 by 256. Click Live and observe the neurons of interest. Then, starting from the dorsal-most side, find a focal plane in which the cells to be ablated are in focus. Using the Regions function, mark a small circular area about one-third the cell size in diameter on each neuron as an ROI.

Set the scan speed to 13.93 seconds per 256 by 256 pixels, which corresponds to a laser dwell time of approximately 200 microseconds per pixel, or 200 microseconds per half micron. Also, set the iteration cycle to 4 repetitions. Click Start Experiment to execute the time series and bleaching functions. Compare the image before ablation to the image after ablation in the time series cycle, to ensure that the fluorescence in the targeted cells is gone.

Next, move the focal plane slightly deeper, and choose the next focal plane where unablated cells appear. Carry out the bleaching function as just demonstrated, and repeat the process until all the cells in the neural structure of interest have been ablated. Following ablation, check the cells for abolished fluorescence, and repeat the laser irradiation if necessary.