To begin, prepare the experiment and material required for zebrafish mounting and positioning. Under a stereo microscope, observe the paralyzed zebrafish larva to verify its movement has stopped. Evaluate the larval health visually by checking its heartbeat.
Then, using a transfer pipette, place a single larval zebrafish into the 1.5-milliliter micro centrifuge tube containing agarose gel. Pour the agarose gel into the Petri dish to make a one-to two-millimeter coat. Transfer the larva from the micro centrifuge tube to the Petri dish using a transfer pipette.
Ensure to place the larva in the center of the dish. Using forceps, position the larva in the desired orientation so that the head and tail are flat. Then, using forceps, rotate the larva to level both eyes.
After the alignment is complete, wait until the agarose gel has solidified before filling the Petri dish with egg water. Place the dish with the embedded larva on the microscope stage for image acquisition. Turn on the microscope and locate the larva at the center of the field of view.
Using the image acquisition software, set the imaging parameters. If the image is saturated, reduce the laser power. Next, find the brain of the larva by moving the stage, and determine its thickness using Live View mode in the software by changing the focal planes manually up and down.
Set the lower and upper limits of the volume. Then proceed with the image acquisition for the set field of view. For volumetric structural imaging, acquire a 3D image of the entire brain by obtaining 2D images of each Z-plane sequentially.
For functional imaging of a single Z-plane, acquire time series images of the neuronal activity of the brain at a certain depth.
