This protocol allows imaging mouse embryos as they develop. This enables studying early cardiogenesis in detail. Like taking still images of fake embryos, live imaging gives full access to study dynamic process in embryo development.
The skills required for life imaging are hard to grasp from text only publication. Watching another person doing it is essential to learn. To begin, cut one centimeter long tungsten wire pieces and sharpen them to 0.02 to 0.05 millimeters in diameter by submerging the tip of the wire in a saturated sodium nitrate solution.
To prepare elbowed glass capillaries, place the midpoint of a standard one millimeter glass capillary over a bunsen lighter for three to six seconds, pulling slowly from both ends until the capillary turns thin and flexible. Then, break the capillary in half and heat it brief briefly to produce 90 degree bend two centimeters from the tip. Saw a piece of polymethyl methacrylate measuring approximately seven millimeters long, four millimeters wide, and two millimeters thick.
Hold the methacrylate piece using a bench vice, then drill customized holes transversely through the whole piece. Rotate the drill slowly while applying low but constant pressure. Use a fine file to smooth the holder's edge and adjust its size.
Additionally, create a mild slope on the edges of the holder to facilitate embryo positioning. Place the finished holder in a dish with distilled water to rinse it. Under the stereo microscope, check the holder to see if the holes contain drilling dust.
If dust is present, use tungsten needles to remove it. To sterilize the holder, place it in a conical tube full of distilled water and sonicate for 20 minutes with a minimum of 20 watt power output. Extract the uterus from a euthanized embryonic day 6.75 to 7.5 pregnant mouse and place it on a dry and clean paper wipe.
Then, cut the uterus, inserting the tip of fine scissors from the mesometrial side, and sliding the blade along to expose the decidui, and transfer them to the dissection medium. Dissect the embryos, keeping the ectoplacental cone intact. Then, peel off the Reichter's membrane, leaving part of it on the extra embryonic region.
To keep the dissection medium warm, replace it every 10 minutes. Moreover, dissect decidui in groups of three to four while keeping the rest in a dissection medium placed in 37 degrees Celsius bath. Immediately place the dissected embryos in a culture medium.
Select the embryos with the desired fluorescence features using a fluorescence stereo microscope and place them in the tube containing the culture medium in the cell culture incubator to recover for two hours before imaging. After turning on all microscope components, including computer, software and lasers, turn on the carbon dioxide controller and set it to 7%Place a drop of high vacuum silicon grease on a 35 millimeter dish with a glass cover slip bottom. Place the holder on top of the drop and press gently to immobilize it.
For embryo mounting, transfer two to three embryos from the incubator to the dish with a holder. Using a pair of forceps and a tapered tungsten needle, set the embryos in the holes. Use the needle to hook the embryo by the ectoplacental cone and insert it into a size matching hole.
Carefully move the dish containing the embryos and the holder to the microscope plate. Lower the objective until a liquid meniscus is formed at the medium objective interface. After placing the embryo in focus, mount the incubation chamber and seal it around the objective using tape.
Using a live capture at the microscope control software, adjust the laser output and gain levels. Then, cover the medium with paraffin oil by dripping it slowly on the objective. Set up a time lapse recording.
Set a five to 10 minutes interval with three to five micron Z space between stacks. Leave a blank space on top of the Z-Stack to anticipate embryo growth. A minimum of 50 microns, adding 30 microns for every hour the acquisition will not be supervised.
Enable the auto save option to allow supervision of the acquisition from a computer to allow the adjustment of the Z-Stack size if needed, to fit the area of interest within focus. Live whole body embryo heart development by multiphoton imaging is shown. At embryonic day 7.5, venous fluorescence is present throughout the neural ectoderm, with a few positive nuclei at the splanchnic and extra embryonic mesoderm.
After four hours, endothelial progenitors activate venous and assemble to form endocardial tube and underlying aortas, which become enclosed structures after seven hours. Be careful while removing the Reichter's membrane. It can be really stuck to the embryo, especially on prevacillation stages.
During removal, try to pinch the membrane by the extra embryonic region.
