従来のUVレーザー共焦点顕微鏡を用いて細胞ストレスと死のトリガ

The overall goal of this cell ablation approach is to selectively stress or kill individual cells in cell culture or in living animals. This method allows researchers to study the fate of a single dying cell and the response of cells, such as microglia and astrocytes to the surrounding ablation site. The main advantage of this technique is the precise targeting of individual cells, and that this can be achieved with a regular confocal microscope that is equipped with a 405 nanometer laser.

We use zebrafish as a model system to study neurodegenerative diseases such as Motor Neuron Disease or Frontotemporal Dementia. Visualizing the death of a motor neuron and the subsequent consequences provides an excellent opportunity to better understand the dynamic processes involved in disease. Our approach to inflict cellular stress in a dose dependent way allows detailed characterization of cellular interactions.

For example, microglia clearance after nueronal death. While this method provides insights into the disease mechanisms of a living organism, it can also be applied similarly in isolated cells in culture. After setting up male and female transgenic lines for mating according to the text protocol, use a plastic tea strainer to collect embryos after successful spawning by straining the tank water.

Then use egg water to rinse the eggs and transfer them into egg water in a Petri dish. Examine the embryos under a light microscope to determine fertilization. Then incubate fertilized eggs at 28 degrees Celsius.

When the embryos reach two to five days post fertilization or DPF, place them under a fluorescence compound microscope and screen the embryos for appropriate fluorophore expression. Then separate brightly labeled fish into a fresh dish with egg water for later embedding. To embed zebrafish in agarose, prepare an anesthesia solution by adding four grams per liter of MS 222 dropwise to a Petri dish containing egg water.

Prepare a stock of low melting agarose and egg water and aliquot it into 1.5 milliliter microcentrifuge tubes. Place an aliquot into a heat block at 38 to 40 degrees Celsius and let it equilibrate. For imaging times of longer than four hours, pipette approximately 300 microliters of agarose along the inner circle of a glass bottomed dish to prepare a donut shaped circle with a small opening in the center.

To mount the zebrafish in agarose for microscopy, after anesthetizing the fish according to the text protocol, use an adjustable pipette to draw up a larva and let it sink to the bottom of the tip. Drop the larva in a minimal volume of liquid into preheated agarose. Then draw up the fish with agarose and quickly dispense it into a previously prepared, glass bottomed 35 millimeter dish.

Under a dissection microscope, use a standard paintbrush to position the animal or multiple animals within the agarose on their sides so that the body and tail are flat. Allow the embedded fish to sit for 10 to 15 minutes until the agarose is firmly set. Then use approximately two milliliters of egg water with tricaine to carefully top up the 35 millimeter Petri dish.

Place the Petri dish with an embedded larva on the confocal microscope stage and using bright field illumination and 40x magnification, focus on the dorsal side of the animal's spinal cord. Switch to an appropriate fluorescence setting and visualize the structure of interest to confirm that all imaging parameters are as needed for subsequent ablation. To determine the thickness of the structure for UV laser ablation, use the Z drive to verify the top and bottom of the structure of interest by manually focusing up and down.

Manually record the Z plane that will be ablated. For example, the center of the cell. To carry out laser ablation, start the FRAP Wizard by clicking on the drop down menu at the top of the software menu.

From the new window, determine the image parameters for the ablation approach by selecting the format, scan speed, and averaging. If the Z plane for ablation hasn't already been selected, press the Live button and focus through the specimen until the fluorescent structure or the desired Z plane to be ablated is in focus. Once the general image parameters are set, access the Bleach step to control the specific ablation components.

Then engage the 405 nanometer laser by activating it for the bleaching procedure. Next use the Zoom In option to maximize the bleaching intensity at the selected ROI by reducing the scan field, therefore maximizing dwell time. Select one or multiple ROIs for the ablation by using any of the drawing tools in the Image Acquisition window.

Target the axon hillock, for example, with the circular drawing tool of approximately four to eight micrometers. After establishing the ROI, select the Time Course button and confirm the number of cycles the ROIs will be scanned and ablated. Choose the Pre-Bleach and Post-Bleach frames as desired to permit an overview of the whole image just before and immediately after the bleaching process.

These settings allow researchers multiple layers of refinement. Through the adjustment of laser power, scan speed, line averaging, size of region of interest, and repetitions, this technique offers a high degree of freedom to cause stress or death in individual cells. After establishing all the necessary ablation parameters, press Run Experiment and monitor the efficiency of the ablation.

Refer to the text protocol for additional details. In this experiment, UV laser ablation was carried out on GFP expressing motor neurons in the spinal cord of transgenic zebrafish. GFP expression driven by motor neuron promoters such as 3 MINX one, IO one or MET allows high resolution visualization of the cell bodies, the main axons, and the peripheral branches extending to the muscles.

Successful ablation of neurons in the spinal cord has been achieved when the fluorescence fades immediately after ablation and never resumes. The specificity of this approach is confirmed as shown here, where a single targeted neuron was converted using the photoconvertible photofluro Kaede that switches its emission from green to red after exposure to UV light. To target cells with different intensities, multiple layers of fine tuning are available by adjusting the laser power, the scan speed, the line averaging, the size of the ROI to be ablated, and the repetitions.

As shown here, motor neurons with long axonal projections that were ablated at the soma with lower UV laser intensities revealed characteristic blebbing that continued from the soma along the axon over time. While attempting this procedure it is important to remember that each confocal setup is unique, and that the laser power output for each instrument is specific and may differ. Following the procedure, characteristic features of apoptotic cell death can be seen in the ablated neurons.

Apoptotic markers such as XN 5 or morphological changes like somal degeneration or external blebbing can be reproducibly detected. Additional testing can be performed to answer further questions such as how swimming performance is affected by ablation of single or multiple neurons in the spinal cord. By crossing different transgenic zebrafish lines, researchers can investigate the response of other cells to laser-induced cell destruction.

These methods provide a unique experimental platform for understanding the precise molecular mechanisms of a disease such as MND in vivo. After watching this video, you should have a good understanding of how to prepare zebrafish for confocal microscopy and how to perform single cell UV laser ablation.