Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo

The scope of this research is to develop a novel tool that can measure tissue mechanics of a live embryo. Using this tool, we want to understand how tissue mechanics evolves when the embryo is experiencing neural tube closure. This can help us understand the mechanical regulations in embryonic development.

To measure tissue stiffness, current methods such as atomic force microscopy, nano-indention, and a micropipette aspiration, need to contact sample physically and apply force to deform it. This is a highly challenging for measuring tissue mechanics of live embryo in situ. Here we employed an optical technology named Brillouin microscopy.

Compared to other technologies, Brillouin microscopy only uses a focused beam to measure tissue mechanics. So, this is a non-contact and a noninvasive method and has some macro-spatial resolution. This allow us to capture time lapse mechanical image of a live embryo in situ In the future, we want to understand the role of tissue mechanics in embryonic development thoroughly.

In specific, we want to study how genetic factors, biochemical signaling pathway, and tissue mechanics coordinate with each other in morphogenesis. This could provide us with new insight in the prevention of birth disease such as neural tube defects. To begin, cut the filter paper into a rectangular shape of approximately 17 by 20 millimeters and remove the four corners.

Then create approximately 7 by 10 millimeters of hollow center in the filter paper. Attach a commercially available ring to a flexible film layer and create a hollow center in the flexible film layer. Place a prepared ring into a 35-millimeter Petri dish.

After pre-culturing, retrieve the egg from the incubator and place it on its long axis on the egg carton tray. Clean the entire surface of the egg shell with 70%ethanol, and allow it to rest for 15 minutes. Hold the egg on its short axis and crack it at the bottom.

Open the egg over a clean 100-millimeter Petri dish to extract its contents. Using a pipette transfer approximately 10 milliliters of the thin albumen into a 15 milliliter tube for ex ovo culture. Fill the center well of the culture dish with thin albumen.

Using tissue paper gently remove the thick albumen attached to the vitelline membrane of the embryo. Now carefully affix the filter paper to the vitelline membrane, ensuring the body axis of the embryo aligns with the center rectangle on the filter paper. Cut the membrane surrounding the filter paper with scissors.

Using tweezers, gently pull the isolated filter paper away from the yolk in an oblique direction. Flip the filter paper upside down to position the embryo with its dorsal side down. Carefully immerse the entire filter paper from one side of the long axis into the 100-millimeter Petri dish containing WASH medium.

Using a clean pipette, gently spray the WASH medium parallel to the filter paper to remove any residual yolk. Next, carefully remove the filter paper from the WASH medium and use tissue paper to absorb any excess medium from the edges of the embryo. Place a filter paper with the embryo onto the culture dish ensuring that the dorsal side of the embryo is facing downward.

Transfer the culture dish to the onstage incubator for ex ovo culture. Build a culture dishes with a warm WASH medium. When the embryo reaches a desired developmental stage, transfer a filter paper with the embryo to the culture dish filled with the WASH medium.

Place the culture dish into the onstage incubator of the Brillouin microscope. For the calibration process, measure the Brillouin signal of water. Then measure the Brillouin signal of methanol.

Under bright field imaging with a four times objective lens, adjust the laser illumination point to the second pair of somites, then switch to a forty times objective lens and perform fine adjustment of the laser point to the middle of the neural tube. Unblock the laser beam and adjust the focal plane. Scan the embryo using a two-dimensional translational stage and acquire a Brillouin image of the region of interest.

After completing the scan, carefully remove the filter paper with the embryo. Use tissue paper to absorb any excess WASH medium from the embryo. Place the embryo back into the culture dish filled with thin albumen for continuous culturing.

For Brillouin image reconstruction, load the water and methanol signal data. Click set region and select the region with four dots for calculating calibration parameters. Save the calibration results.

Load the embryo scanning data and click start for the processing of the parameters. Finally, reconstruct the 2D Brillouin image based on the Brillouin shifts of all pixels. Brillouin shift in the estimated longitudinal modulus of the neural plate against somite number and incubation time showed an increase in Brillouin shift of the tissue during neural tube closure.