Selective Single-Neuron Laser Ablation in a Zebrafish Larva Using a Confocal Microscope

Take a preheated agarose solution containing an anesthetized transgenic zebrafish larva expressing a fluorescent protein in spinal motor neurons. 

Transfer the larva to a glass dish with an agarose ring.

Using a microscope, turn the larva onto its side. Then, allow the agarose to solidify, immobilizing the larva.

Add buffer containing anesthetic to maintain anesthesia.

Place the dish on the confocal microscope stage and use bright-field imaging to locate the dorsal spinal cord region.

Switch to fluorescence mode to visualize fluorescent motor neurons. 

Determine the central z-plane of the cell soma for precise ablation targeting. 

Configure imaging parameters for high-resolution acquisition while minimizing photodamage. 

Once the desired z plane is in focus, perform ablation on the target neuron region using a focused ultraviolet laser. 

Laser energy disrupts cellular structures, causing fluorescence loss and cell death. Irreversible fluorescence loss confirms precise ablation without affecting surrounding cells.

Use an adjustable pipette to draw up a larva and let it sink to the bottom of the tip. Drop the larva in a minimal volume of liquid into preheated agarose, then draw up the fish with agarose and quickly dispense it into a previously prepared glass bottom 35-millimeter dish. Under a dissection microscope, use a standard paintbrush to position the animal, or multiple animals, within the agarose on their sides, so that the body and tail are flat.

Allow the embedded fish to sit for 10 to 15 minutes until the agarose is firmly set. Then, use approximately two milliliters of egg water with trikane to carefully top up the 35-millimeter petri dish. Place the petri dish with an embedded larva on the confocal microscope stage, and using Brightfield illumination and 40X magnification, focus on the dorsal side of the animal spinal cord. Switch to an appropriate fluorescent setting, and visualize the structure of interest to confirm that all imaging parameters are as needed for subsequent ablation.

To determine the thickness of the structure for UV laser ablation, use the Z drive to verify the top and bottom of the structure of interest by manually focusing up and down. Manually record the Z plane that will be ablated, for example, the center of the cell. To carry out laser ablation, start the FRAP wizard by clicking on the dropdown menu at the top of the software menu.

From the new window, determine the image parameters for the ablation approach by selecting the format, scan speed, and averaging. If the Z plane for ablation hasn't already been selected, press the live button and focus through the specimen until the fluorescent structure, or the desired Z plane to be ablated is in focus. Once the general image parameters are set, access the bleach step to control the specific ablation components, then engage the 405-nanometer laser by activating it for the bleaching procedure.

Next, use the zoom-in option to maximize the bleaching intensity at the selected ROI by reducing the scan field, therefore maximizing dwell time. Select one or multiple ROIs for the ablation by using any of the drawing tools in the image acquisition window. Target the axon hillock, for example, with the circular drawing tool of approximately 4 to 8 micrometers.

After establishing the ROI, select the time course button and confirm the number of cycles the ROIs will be scanned and ablated. Choose the pre-bleach and post-bleach frames as desired to permit an overview of the whole image just before, and immediately after the bleaching process.
 
These settings allow researchers multiple layers of refinement. Through the adjustment of laser power, scan speed, line averaging, size of region of interest, and repetitions, this technique offers a high degree of freedom to cause stress or death in individual cells.

After establishing all the necessary ablation parameters, press Run experiment and monitor the efficiency of the ablation. Refer to the text protocol for additional details.