Visualizing Neural Crest Cell Migration in a Zebrafish Embryo Using Multi-Photon Time Lapse Imaging

Begin with a live, dechorionated transgenic zebrafish embryo embedded in low-melting-point agarose and placed in a chamber filled with embryo media.

The embryo expresses enhanced green fluorescent protein (EGFP)-labeled neural crest cells.

Position the chamber on the microscope stage. Using bright-field illumination, adjust the objective to focus and center the embryo in the field of view.

Switch to the water-immersion objective and refocus on the embryo.

Adjust the imaging parameters to acquire Z-stack images from the lateral to the medial edge, capturing the width of the eye.

Turn off bright-field illumination, cover the stage, and initiate time-lapse imaging.

Refill the chamber with media as needed during imaging.

Under laser illumination, the neural crest cells fluoresce.

Visualize the migration of these cells from the neural tube into the craniofacial regions, around the developing eye to populate the periocular mesenchyme and frontonasal process, and ventrally to form the pharyngeal arches.

After placing the entire setup onto the stage of the microscope, according to the text protocol, use the five times objective to locate the embryo. Then manually raise the stage to the highest position, and use the fine focus to position the embryo in the middle of the microscope range.

Manually lower the stage, and change the five times objective to the 25 times water immersion objective. Carefully raise the stage to bring the embryo back into focus. In the software, click on the xyzt mode for obtaining multiple images at time or t intervals in the xy plane over a depth of z.

Use the epifluorescence or brightfield view to find the depth of focus in the area of interest, which will demarcate the z-stack. For these experiments, the lateral edge of the eye is the beginning of the z-stack. When focused here in the software, click the Begin button. After focusing down to the midline of the embryo, which is the end of the z-stack, click the End button.

A critical step is to make sure that the z step encompasses the area of interest. In our case, we want to capture the width of the eye from the lateral to medial edges.

Click on the menu for adjusting the acquisition time and imaging frequency. For adequate recovery of the fluorophore and survival of the embryo, allow for a ratio of at least 1 to 3 between z-stack acquisition when laser power is on, and recovery time when laser power is off. With appropriate time for embryo recovery, set the time between z-stacks and the designated window. Then set the total length of time for the experiment in the appropriate window.

Now turn on the live image setting to make final adjustments to the laser settings. Adjust laser transmission, gain, and offset slider bars within the software to optimize the fluorescence image. Also, adjust the orientation of the embryo as needed depending on the length of the experiment, anticipated growth of the embryo, et cetera. Make sure that the area of interest remains within the frame through the duration of the experiment.

During time-lapse acquisition, turn off the epifluorescence light source. Cover the stage with the laser safety box, which is adequate for protection against background light. Then press Start.

Access the open bath chamber through the sliding doors on the laser safety box to refill it with time-lapse embryo medium every 8 to 12 hours. Following imaging acquisition, open the files in the image processing software. Highlight the correct image series.

In the software, choose the Process menu. Click on 3D Deconvolution and apply to deconvolve each z-stack. Import individual TIFF files into video processing software. Then select all TIFF files and drag them into the video editor. Adjust the length of each image within the video to 0.1 seconds. Finally, export the video as a MOV or MP4 file.