Two-Photon Laser-Mediated Ablation of Osteoblasts in Developing Zebrafish Larvae

Our research focuses on bone regeneration. We try to understand the mechanisms that drive bone regeneration. In particular, how the immune system is involved in the restoration of bone.

The immune response is essential for bone homeostasis and regeneration. The absence or excess of an inflammatory response after injury impairs the regeneration process. For that reason, immunomodulation must be tightly controlled to allow for successful regeneration.

We have shown that macrophages, which are innate immune cells, are recruited to bone injuries and that immunosuppression impair bone regeneration. To begin, set up parental zebrafish with the desired single transgenes in standard mating cages the evening before mating. Using a divider in the mating cage, keep males and females separated until mating.

Remove the divider in the morning of the next day to initiate mating. At noon at the latest, remove parental zebrafish from the breeding tanks. Strain the water from the mating cage through a sieve to collect the embryos.

Then place the embryos in a 100 millimeter diameter dish containing E3 media and keep them in an incubator at 28 degrees Celsius. Afterward, remove unfertilized eggs and dead embryos from the dish using a plastic pipette and a stereomicroscope. Using a three milliliter plastic Pasteur pipette, transfer the four-day post-fertilization larvae with reporter transgene expression to a glass bottom microwell dish.

With the same pipette, remove the excess E3 media. Now, take the melted low melting agarose from the water bath or thermoblock. Add a cooling drop of low melting agarose to the dish, covering the glass bottom where the larvae were positioned.

Place the larvae into the desired lateral position before the agarose solidifies. Once the agarose solidifies, add E3 media containing a maximum of 0.02%MS222 to the dish to avoid drying of the agarose. To begin, embed the transgenic larva in low melting agarose in a glass bottom microwell dish.

Place the dish onto the microscopic stage at the inverted microscope. Position and focus the desired region for ablation in the center of the field of view. Choose the Z-stack settings to image the entire breadth of the opercle.

Image the opercle area of interest before ablation. Then draw a circular region with a diameter of 25 micrometers in a selected 2D plane. Focus on the opercle using the region of interest or ROI tool.

To perform the ablation, apply ablation power measured without objective to 450 milliwatts for a duration of 10 seconds. Expose the selected area to the two-photon laser. After ablation, confirm the ablation structurally by imaging the same Z-stack with the same settings as used prior to ablation.

Using forceps or a dissection needle, remove the larvae carefully from the low melting agarose First, remove a layer of agarose covering the larvae. Second, remove the agarose in direct contact with the larvae. Place the larvae back in a dish with pure E3 media.

At four or six hours post-lesion, re-image the opercle area using agarose-embedded larvae at the same settings on the same microscope. After processing the images in Fiji, quantify cells with a nuclear label or by measuring the area in case cells are overlapping. To quantify the area, create a mask encompassing the macrophages.

Use the Image, Adjust, Threshold Tool, Create ROI, and analyze the particles. Then measure the area. Generate graphs plotting the measurement results using suitable software.

A significant accumulation of macrophages in the ablated opercle region was detected six hours post-ablation in six days post-fertilization larvae. In larvae four days post-fertilization, the number of macrophages increased at four hours post-lesion. Macrophage number and area increased at four hours post-lesion compared to the unablated state.