Using this protocol, any lab that has a standard confocal microscope equipped with a 405 nanometer laser can perform hair cell progenitor laser ablations and monitor their regeneration. Unlike electro-ablation, this technique limits damages surrounding cells and is more accessible than a powerful pulsed UV laser setup. Confocal imaging can also be performed immediately before and after ablation.

This technique enables us to better understand the regenerative behavior of sensory progenitors, which may help in the development of therapies for human hearing loss. For mounting, first pipette three to four anesthetize larvae into a small droplet of E3 tricaine solution into the center of the 35 millimeter dish, with a 14 millimeter number 1.5 cover slip bottom. Remove the excess solution so that the larvae remain in a small droplet just large enough to contain them.

And place the dish on the stage of a binocular stereo microscope. Manipulate the zoom and focus so that all of the larvae are in the field of view and use a transfer pipette to add a thin layer of agro-solution onto the cover slip. Remove the excess agros until the liquid just fills the well at the bottom of the dish, taking care not to aspirate any larvae.

And use a hair knife to quickly orient the larvae in the agro-solution with the rostral side facing left. Gently press the larvae down against the glass, such that the larvae lie in profile with their right sides facing down. After about 60 seconds, the agros will start to solidify and the larvae will not be able to be reoriented.

After five minutes, use a transfer pipette to fill the dish halfway with E3 supplemented with 1X tricaine. To locate prospective targets, turn on the power to the laser scanning confocal microscopy system and initialize the laser through the integrated imaging software. Select the 63X Plan-Apochromat oil immersion objective and apply immersion oil to the lens.

Secure the dish in a circular stage insert with the rostral aspect of the larvae facing to the left. Using bright field or differential interference contrast illumination, select one of the mounted larvae for imaging and use the focus knob to bring the skin on the side of the fish closest to the cover slip into focus. Switch to epifluorescent illumination in the GFP channel and locate the posterior lateral line by GFP expression along the horizontal myoseptum.

Rings of fluorescent cells are indicative of the neuromast mantle cells and elongated strands of cells are the interneuromast cells. Beginning with the first migrating primordium neuromast, use the stage control joystick to visually scan caudally along the horizontal myoseptum. Following the string of interneuromast cells until the region between the third and fourth migrating primordium neuromasts is reached.

If several larvae are to be imaged, select position to set the first stage position. After cell bodies in the L3, L4 region have been identified, switch to the acquisition mode and use an appropriate laser to activate the GFP imaging track. To add a transmitted light channel to the activated laser track, click the T-PMT box in the imaging setup dropdown menu.

To image ET20 larvae, select the 488 nanometer laser, set the laser power to 6%the pinhole size to one area unit equivalent and the digital gain to 750. Adjust the gains such that the cell bodies are saturated to capture otherwise dim projections and filopodia. And set the frame size to 1, 024 by 1, 024 pixels, the averaging to two and the digital zoom to 0.7.

Check the Z stack box to bring up the Z position dropdown menu. While fast scanning, focus out until the interneuromast cells are just out of focus and set the first slice. Focus through the sample until the interneuromast cells are once again out of focus and set the last slice.

Then click stop and click start experiment to capture a pre-ablation Z stack. If the stage positions have been added, inactivate the positions option so that only the current position is imaged and save the file once it has been captured. For laser ablation of the targeted cell bodies, click show all tools in the acquisition interface and in the imaging setup menu, select add a new track.

Click dye and select DAPI. Under channels, select 405 for the laser setting and increase the laser power to 75%Unclick the DAPI channel to turn off the laser while scanning for candidate cell bodies for ablation. Click live and with the body of an interneuromast cell centered in the field of view, zoom into the scanning frame to 20 to 22X.

Stop the live scanning as soon as the cell body fills the field of view. Check the 405 nanometer laser shutter box to activate the track and set a timer for 45 seconds. Then, activate continuous scanning and start the timer.

Immediately stopping the scanning at 45 seconds. 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 pre-ablation 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.

In this representative experiment, the region of the lateral line located between the third and fourth migrating primordium neuromasts was identified and pre-ablation images were captured. Post-ablation scanning confirmed that no cell bodies remained in the ablated region. Leaving a gap between the elongated projections of the adjacent interneuromast cells.

Analysis of the transmitted light photomultiplier tube channel after ablation, reveals damaged and dying cells. Marked by swollen and irregularly shaped nuclei and a granular appearance. The recruitment of large amoeboid cells that are likely macrophages may also be observed.

In this experiment, the ablation of several cells in the double transgenic larvae created sizable gaps in the interneuromast cells string, but had little or no effect on the lateral line nerve. Following laser ablation, gap sizes can be measured. Ranging from just a few microns up to 100 microns, depending upon the width that the individual neuromast cells and how many cells are selected for ablation.

After ablation, some interneuromast cells recover within the first several hours of imaging. With the probability of gap closure negatively correlating with gap size. Even in interneuromast cells that are unable to recover however, the formation of long projections from neighboring interneuromast cells, which can resemble extending neuronal growth cones can be observed.

It's important to thoroughly inspect the T-PMT channel for damaged cells exhibiting irregularly shaped nuclei and granularity, and to allow sufficient time for these indicators of cell death to become visible. Further analysis of the resulting time-lapse microscopy data, can potentially reveal novel cellular behaviors induced by laser ablation and may guide the development of experiments for identifying regulators of regeneration. This technique has enabled us to study the molecular regulators of interneuromast cell regeneration by providing a rapid and cost effective method for selectively damaging these cells.

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