A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

The overall goal of this technique is to culture chick embryos ex ovo for time-lapse imaging by keeping them fully submerged in a liquid culture medium. This method can help answer key questions in a study of morphogenetic events during early embryogenesis, such as neurulation, gastrulation, somitogenesis, heart and brain formation. The main advantage of this technique is that there is no need for a climate chamber around the microscope.

A layer of light mineral oil prevents evaporation from the culture medium. To begin, prepare 50 milliliters of culture medium by combining 30 milliliters of freshly prepared Pannett-Compton saline with 20 milliliters of thin albumen. Mix the components gently by inverting the tube several times.

Next, place two small stainless steel rings as far apart as possible in a fresh six-centimeter plastic Petri dish, and use a 25-milliliter plastic pipette to fill it with 22 milliliters of culture medium. Make sure the debris accumulated on top of the medium remains in the centrifuge tube. Then, use a plastic transfer pipette to remove any debris or bubbles from the medium in the Petri dish.

Next, fill a 10-centimeter Petri dish with 75 milliliters of Pannett-Compton saline to be used in a later step. After the desired incubation time, remove an egg from the incubator, and let it rest on the bench on its side for one minute to let the embryo come to the top of the yolk. Then, carefully crack the egg into a Petri dish, and soak the edge of a piece of fine tissue paper in the thick albumen.

Use the tissue paper to center the blastoderm by pulling it into position. Next, cut the tip of a transfer pipette, and use it to remove most of the thin and thick albumen from the Petri dish. With most of the thick albumen removed, use a piece of tissue paper to gently wipe away the remaining thick albumen from around the embryo in order to create a window.

Take care that the tissue paper does not attach to the vitelline membrane. Using tweezers, carefully place a filter paper carrier on the vitelline membrane around the embryo so that the longer axis of the elliptical aperture is parallel to the anterior-posterior axis of the embryo. Then, pinch one tip of the nail scissors through the vitelline membrane close to the filter paper carrier, and quickly cut around the whole filter paper carrier.

Using tweezers, lift the filter paper carrier away from the yolk in an oblique direction that is parallel to the anterior-posterior axis of the embryo, then turn the filter paper carrier around to have the ventral side of the embryo on top and submerge it in the Pannett-Compton saline. Immerse the filter paper carrier along the anterior-posterior axis of the embryo in an oblique direction. Next, get rid of all the yolk that is still attached to both the vitelline membrane and the blastoderm by gently flushing it with Pannett-Compton saline from a plastic transfer pipette.

Work quickly to minimize the time the embryo spends in a saline bath. Always point the saline stream away from the embryo, and never squirt directly on it. Remove the filter paper carrier from the Pannett-Compton saline along the anterior-posterior axis of the embryo in an oblique direction, and get rid of the excess liquid by dabbing the edge of the filter paper carrier on a tissue paper.

Hold the filter paper horizontally, and with the ventral side of the embryo facing up, use a second pair of tweezers to place a second filter paper carrier on top of the first one so that their apertures line up exactly, sandwiching the embryo between them. Immediately submerge the filter paper carrier sandwich into the culture medium in an oblique direction along the anterior-posterior axis of the embryo. Place it in the area spanning the space between the two steel rings.

Next, fix the filter paper sandwich in place by setting two more steel rings on top of the other two, making sure that the aperture with the embryo remains completely uncovered. Check the medium level, and make sure that the upper spacer is barely submerged in the medium. If necessary, add or remove small amounts of the medium.

The culture is now ready to be placed into the imaging set-up. Carefully place the Petri dish in the center of the temperature controlled oil bath, and stabilize the dish laterally by placing three large stainless steel rings next to the dish in the oil. Make sure that the steel rings touch the sides of the dish.

Then, use a plastic transfer pipette to slowly add approximately six milliliters of light mineral oil on top of the medium so that the medium is covered with a continuous layer of mineral oil. This video shows a complete time-lapse of an embryo in the well-established filter paper carrier culture called EC culture, being cultured ventral side up. This embryo starts out at the seven somite stage and is imaged with a temporal resolution of four minutes.

Later development of the embryo is hindered by strong confinement in the z-direction. In contrast to the EC culture, embryos cultured in the submerged filter paper sandwich set-up are not limited in their three-dimensional expansion. They develop a proper cranial and cervical flexure and a functional blood circulation.

The development of the embryo was imaged consecutively over 46 hours, but after approximately 30 hours, the focus on the segmenting mesoderm got lost due to the overall thickening and turning of the embryonic tissue. This embryo is being cultured dorsal side up. The lower panel shows the local swelling of the neural tube, leading to the regionalization of the forebrain, midbrain, and hindbrain, which will divide into the five subregions of the embryonic brain at later stages.

The embryo dies at the 28 somite stage, also referred to as Hamburger Hamilton stage 16. Once mastered, it takes about 10 to 15 minutes to bring an embryo into the submerged filter paper sandwich. Before applying mineral oil to the surface of the culture medium, the embryo is still accessible to micro-manipulations, such as microsurgery or bead implantations.

In combination with the emergent objectives, the submerged filter paper sandwich will be very useful for laser-based light fluorescent imaging, including light sheet microscopy.