To begin, cut the filter paper into a rectangular shape of approximately 17 by 20 millimeters and remove the four corners. Then, create approximately seven 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 eggshell 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 albumin into a 15-milliliter tube for ex ovo culture.
Fill the center well of the culture dish with thin albumin. Using tissue paper, gently remove the thick albumin 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 yoke 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 the 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. Fill the culture dishes with a warm wash medium.
When the embryo reaches a desired developmental stage, transfer the filter paper with the embryo to the culture dish filled with a 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 40 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 albumin 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 2-D 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.
