This protocol is the first complete laser ablation protocol on early kelp embryo. The procedure offers a reliable approach on investigating cell fate and interaction during embryogenesis in brown algae. Local cellular laser ablation allows for temporal and special ablation with a high level of precision and tissue on cells.
This approach is a promising method for studying cell fates and interaction in the early embryo of brown algae. It can be applied to other algae systems with few changes. Begin by imaging the entire Petri dish to locate the embryos for selection during the ablation step and monitor the subsequent development of the selected embryos.
Start the tile scan. Record the position of the four cardinal points of the Petri dish. Enter the parameters as defined in the text manuscript and acquire transmitted or fluorescent images of the whole Petri dish at low resolution.
Save the tile scan image and keep it open in the image acquisition software window. Change the objective without removing the Petri dish. Find embryos of interest on the scan.
To calibrate the laser, open the laser driver and image acquisition software package. Then initialize the laser path. Synchronize both software packages by clicking on start acquisition in the laser driver software package.
Set up the parameters for laser ablation and click on live. Select an empty area on the dish and lower the stage level to 20 micrometers below the top surface of the Petri dish to focus on the glass bottom. To find an area of interest by clicking on choose AOI button and clicking on the edges of the image in the UV laser driver software package.
Click on start calibration and select manual calibration to set the ablation laser and imaging laser trajectories. Ensure that all shutters are open. Select a laser power high enough to see a black dot in the center of the live image corresponding to the hole in the glass cover slip.
Click on the central black dot with the mouse cursor and select 18 additional dots proposed by the software to complete the alignment procedure. Click on the click and fire mode. Check the calibration on the same cover slip and click on the glass layer where the laser impact is visible.
Select an embryo of interest on the tile scan and move the stage by clicking on it. Start the time series. Test time lapse parameters in live and adjust if necessary.
Start a time lapse recording in the image acquisition software at the maximum speed. Adjust the zoom to focus on the area of interest and visualize the whole embryo. To follow the embryo during the recording of ablation, click on the button, acquire latest image.
Set the parameters to 45%laser transmission corresponding to a maximum of 40 microwatts and one millisecond pulse time duration. Apply the damaging irradiation on the embryo cells of interest by using the laser driver software click and fire function. Under 688 nanometers, monitor the autofluorescent chloroplasts ejection from the cytoplasm.
If cell contents remain in the cell, use the click and fire function once more to increase the size of the breach in the cell. Repeat this process until most of the cell contents are released, keeping the number of shots to a minimum. Stop the time lapse recording after the embryo has stabilized and no further intercellular movement can be detected.
Update the annotation on the tile scan image, overwrite the existing tile scan, and save the image. Determine the survival rate by monitoring the number of embryos that develop after laser ablation and compare them to those that die. Determine the growth delay by measuring the length of the laser shot embryos every day and comparing it to intact embryos.
Find the adjacent damage by monitoring the reaction of cells adjoining the ablated cells. Targeted cells of interest were the most apical cell, the most basal cell, and the median cells. After tile scanning the entire Petri dish, an embryo of interest was identified as a suitable candidate for laser shooting.
The cell released its contents when shot with a pulse UV laser beam at 45%power, while a cell's adjacent to the irradiated cells expanded into the intercellular space. The position of the irradiated embryos was recorded every 24 hours for 10 days. Most of the irradiated embryos continued to develop but showed growth alteration.
In contrast, embryos tested with other laser parameters quickly showed signs of severe stress such as cell bleaching, fading, or shape changes. Almost all embryos shot in this way died within five days after the experiment. Some parameters, such as image format or zoom should not be changed after calibration.
Monitoring during and after laser ablation is required to have insight into what happened. Laser ablation is paving the way to new discoveries in the developmental study of brown algae and more profound understanding of interactions of different metabolites or components of their cells.
