Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula

Within the embryos, cells interact with each other, exchanging chemical and mechanical information. One efficient way of probing these interactions is to kill some cells and monitor the consequences on neighboring cells. The method we describe allows us to accurately ablate cells, even within the embryo, without damaging neighboring tissues.

Prepare the two-photon microscope by setting the first laser to the ablation wavelength 820 nanometers, and the second laser to the mCherry imaging wavelength 1, 160 nanometers. Using movable mirrors on the optical path, align the two laser beams at the scan heads entry and exit, then measure the maximum power of the first laser at 820 nanometers. Place the power meter under the objective, close the black chamber, and set first laser power to 100%Start data collection on the Power meter, open the laser shutter then measure the output power and compute the percentage of laser power needed to reach 300 milliwatts.

Set the first laser to the GFP excitation wavelength of 920 nanometers and power to 7%Adjust the second laser power to 15%Activate the PMT detectors for green and red lines, and set green and red line PMT sensitivity to 65. Adjust the field-of-view to 400 by 400 micrometers, image resolution to 512 by 512 pixels, and scanning frequency to 800 hertz. Select 3D time lapse imaging mode, then create a folder and activate Autosave to save data after each acquisition.

Assemble the heating chamber and set it to 28 degrees Celsius. Wait at least 10 minutes for the chamber and objective to warm. Under a fluorescence stereomicroscope, identify embryos at 70%epiboly that express GFP.

The, using a plastic Pasteur pipette, transfer three to four selected embryos in the agarose-coated dish and carefully dechorionate them with fine forceps. In a small glass vial, add one milliliter of a solution of penicillin-streptomycin embryo medium containing 2%agarose, and place the vial in a preheated, dry block heater at 42 degrees Celsius. Using a fire-polished glass pipette, transfer a dechorionated embryo to the vial, taking care not to add too much embryo medium to the agarose.

Discard the remaining embryo medium from the pipette. Aspirate the embryo back, along with enough agarose to cover the slide of the glass bottom dish. Blow the agarose with the embryo on the glass slide of the dish, ensuring the embryo is not touching the air or the plastic side of the dish.

Then, fill the chamber around the glass slide with agarose. Using an eyelash, orient the embryo so that the targeted region is at the top. After five minutes, when the agarose is completely set, add a few drops of penicillin-streptomycin embryo medium to the glass slide.

Place the glass bottom dish under the objective in the heated chamber. Immerse the objective in penicillin-streptomycin embryo medium before closing the chamber. Move the slider to set the light path to oculars.

Turn the fluorescence lamp on. Find an embryo and set the focus to its surface. Turn the fluorescence lamp off and set the light path to PMTs before closing the black chamber.

Start live imaging and locate the axial mesoderm. To have a good signal, adjust the laser powers. Use the red channel to move the stage to the very top of the embryo, and assign this position as Z equals zero.

Choose a time step of one minute and a Z-step of two micrometers. Set the first slice at 15 micrometers above the axial mesoderm, and the last slice at 15 micrometers below the axial mesoderm. Record 10 to 15 minutes of a pre-ablation movie.

On live imaging, locate the polster contour, then, using the electro-optic modulator region of interest, or EOM-ROI tool, draw a 20-pixel-large rectangle that spans the width of the polster, and place the rectangle in the middle of the polster. Note the axial position of the highest and lowest planes to be ablated, ensuring that the ROI to does not overlap the yolk cell on any of the planes. Place the stage at the lowest Z-position to be ablated.

Set the first laser wavelength to 820 nanometers and enter the power percentage as calculated earlier to obtain an exit power of 300 milliwatts. Set the imaging frequency to 200 hertz and first laser imaging EOM to zero. After selecting ROI Treat mode, turn off the EOM, and set the treatment to start immediately after zero frame.

Put the imaging mode to Timelapse and deactivate Autosave. After selecting Fast mode in Time step, adjust the number of treatment frames and number of frames to the value corresponding to the targeted depth. Start imaging.

The acquisition should be black, as the shutter to PMT closes during EOM treatment. Next, move up the stage to the next Z-position, and similarly perform ablation. Repeat on every Z-position until the top of the polster is reached.

When the process of ablation is complete, set the first laser to 920 nanometers and adjust to the previously-chosen imaging power. Put the first laser imaging EOM to 100 and opt for the Full field mode. Imaging frequency should be 800 hertz, and EOM should be turned off before examining the whole stack in live mode to check whether every plane has been ablated.

Once ablation has been confirmed, select 3D Time lapse as the imaging mode. Reactivate Autosave, and start recording the post ablation movie for 40 to 60 minutes. In the post-ablation movie, check whether the targeted cells were effectively ablated.

As ablations across the polsters should not harm embryos, the normal morphology of the ablated embryos can be observed at to 24 hours post-fertilization. In the successful outcome of laser ablations, the overlaying tissues were not affected by the ablation of underlying structures. A too-intense treatment, or too little treatment, resulted in failures in laser ablation.

An example of unsuccessful ablation shows cells above the polster being ablated, which was evident by autofluorescent debris in the focal plane above the polster. In another ineffective attempt, cells were bleached but not ablated, and low-fluorescence signals still reveal the intact cell contours. Finally, some cells were not even bleached due to insufficient laser treatment.

The formation of bubbles is due to cavitation induced by a too-intense laser treatment. Z-stacks were captured every minute for 40 minutes in a successful ablation, recording both the cytoplasmic GFP and the nuclear mCherry signals. In addition, nuclei belonging to the anterior half of the polster are marked with a magenta dot and tracked over time, before and after ablation.

As a measure of migration persistence, direction auto-correlation of cells before and after ablation was studied. Cells belonging to the anterior part of the polster displayed a persistent motion before ablation, which drastically decreases after ablation, indicating a loss of collective oriented migration. Properly aligning the lasers and measuring laser power are critical to get reproducible results.

It is also key to check the ablation efficiency on the first post-ablation images. We use laser ablations to question the role of cell-cell interactions in guiding the migration of cells within the embryo. But the procedure could be easily adapted to many other questions where cells or tissues need to be precisely ablated, potentially deep in the organism.