In Vivo Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy

We are developing optical immune systems and computational algorithms to record and analyze the neuroactivity of the entire brain with a high spatial and temporal resolution. Larval zebrafish is an ideal model animal for research thanks to its optical transparency and the availability of diverse genetic tools. The optical image quality may be degraded due to the aberration introduced by the agarose gel used for sample mounting, and the fish may move during the recording, causing motion artifacts in the images or impeding accurate signal extraction from the images.

Publicly available protocols provide only a brief overview of the experimental procedure, leaving substantial parts of the details, such as agarose solidification, precise mounting techniques, and simple positioning. Therefore, an effective and reproducible protocol is needed to acquire high-quality image data with minimal noise and motion. Our protocol provides an optimized and reproducible experimental procedure.

This protocol enables in vivo whole-brain imaging over an extended period and visualizations of the acquired imaging data. The workflow focused on whole-brain imaging, but it can be easily applied to imaging other organs of larval zebrafish. Our goal is to unravel the underlying principles of neural computation.

For that, we'll continue to work on a pipeline that involves large-scale imaging of neuroactivity and structure and computational analysis of such for systematic brain mapping. To begin, prepare the experiment and material required for zebrafish mounting and positioning. Under a stereomicroscope, observe the paralyzed zebrafish larvae 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 microcentrifuge tube containing agarose gel. Pour the agarose gel into the Petri dish to make a 1 to 2-millimeter coat.

Transfer the larvae from the microcentrifuge tube to the Petri dish using a transfer pipette, ensure to place the larvae in the center of the dish. Using forceps, position the larvae in the desired orientation so that the head and tail are flat. Then using forceps, rotate the larvae 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 larvae on the microscope stage for image acquisition. Turn on the microscope and locate the larvae 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 larvae 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. After installing napari and napari-animation, create a new Jupyter notebook file. To visualize images and create movies of the rendered zebrafish brain load the 3D image and open the napari window, then connect the napari-animation plugin.

Next, set the parameters, such as voxel size, colormap, and contrast limits in the layer controls. Adjust the viewer settings in the canvas. Then, to capture the rendered image, press the Capture button in the animation wizard.

To generate movies of the rendered volume, modify the viewer settings and add key frames. After adding key frames, set the number of frames between key frames in the animation wizard. Finally, save the rendered animation.

The volumetric field of view of a larval zebrafish brain expressing pan-neuronal GCaMP7a covered the brain regions of the forebrain, midbrain, and hindbrain. Neuronal cell bodies of the medulla oblongata, cerebellar plate, optic tectum, habenula, and dorsal telencephalon were clearly visible in the whole brain of a larval zebrafish. In 2D functional imaging, neural cell bodies were clearly visible in both the background and the superimposed neural activity.