Monitoring Recovery of Laser-Ablated Interneuromast Cells in Zebrafish Using Confocal Microscopy

Begin with a zebrafish larva containing fluorescently labeled interneuromast cells or INMCs, mounted on a coverslip and placed under a confocal microscope. 

INMCs are progenitor cells with regenerative ability that have been previously subjected to laser ablation.

First, observe the damaged area in fluorescence mode. The absence of fluorescence confirms successful laser ablation. 

Next, use transmitted light imaging mode to examine the damaged area where the cell appears granular and swollen with irregular nuclei, confirming cell death.

The recruitment of macrophages to the site indicates an active cellular response to injury.

Now, switch back to fluorescence mode and capture time-lapse images at regular intervals to monitor recovery. 

During the recovery phase, INMCs extend projections into the gap, facilitating closure and promoting cellular repair. 

Save these time-series images for analysis. The reappearance of distinct fluorescence in the ablated area confirms successful cell recovery.

For imaging of the cell bodies post-ablation, under the Channels menu, unclick the DAPI track to inactivate the ablation laser, and open the acquisition mode menu. Click Zoom and decrease the zoom to 0.7.

To assess the success of the cell ablation, fast-scan the field of view. Using the same settings as those for the preablation imaging, capture and save a post-ablation image. Inspect the transmitted light photomultiplier tube channel image to further confirm the cellular damage. Damaged cells will demonstrate a granular appearance, and the nuclei will frequently swell or appear irregular in shape.

To assess the post-ablation cell body recovery, activate both the stage position and time options for time lapse image capture, and set the time parameters to the appropriate experimental time point and 15 minute intervals. Then start the experiment to acquire images, and save the resulting file when complete.