6. Place individual embryos in conical wells containing 30 μL of
culture medium. Carefully add sterile water into the dedicated
ridges of the microslide. It must not overflow so it does not
dilute the culture medium, and so the lid of the microslide does
not get sealed to its bottom by the capillarity of the water. It is
advised to let embryos recover from dissection for 1 h before
proceeding to imaging.
3.3 Static Culture
and Imaging
of Gastrulating
Embryos
Embryos can develop normally under the microscope for up to
24 h.
1. Pre-warm the incubation chamber to 37
C and equilibrate
CO 2 /O 2 levels.
2. Transfer the microslide from the incubator to the microscope
incubation chamber.
3. Several embryos can be imaged in a single experiment. As they
grow and are not physically attached, it is not practical to use
the automatic positioning program. Rather, position in X, Y,
and determination of optimal Z-stack have to be set up for each
embryo at each time-point. Before starting the time-lapse, one
should ensure optimal positioning of the embryos (Fig. 3). At
that stage of development, anterior-posterior axis can be determined through morphology alone. To visualize gastrulation
EMT, they should lie either on their posterior or lateral side,
which are natural positions due to the ellipsoid embryo shape.
Embryos can be gently flipped in the conical well using coarse
forceps in a closed position, or a plastic tip.
4. It is essential to minimize embryo exposure to light (see Note 6).
3.4 Image Analysis
Imaging data should be stored on two separate servers as backup.
Images are viewed and analyzed using an image analysis program
installed on a powerful workstation. Confocal and two photon
generated images are 3D reconstructed, projected on the Z axis,
and their brightness and contrast enhanced (Fig. 4). For time lapses
acquisitions, embryos must be registered using a drift correction
tool. Embryos are highlighted on each Z slice to generate a 3D
virtual embryo. The same embryo is registered across time by
matching its center of gravity. Individual cells are then segmented
through manually highlighting cell membranes along the Z slices
using a graphic tablet. The cells are then tracked over time using the
imaging program tracking algorithm. Tracking and cell shape data
can then be funneled through a dedicated python script [14] for
statistical analysis.
Ex vivo Imaging of EMT in the Early Mouse Embryo
141
culture medium. Carefully add sterile water into the dedicated
ridges of the microslide. It must not overflow so it does not
dilute the culture medium, and so the lid of the microslide does
not get sealed to its bottom by the capillarity of the water. It is
advised to let embryos recover from dissection for 1 h before
proceeding to imaging.
3.3 Static Culture
and Imaging
of Gastrulating
Embryos
Embryos can develop normally under the microscope for up to
24 h.
1. Pre-warm the incubation chamber to 37
C and equilibrate
CO 2 /O 2 levels.
2. Transfer the microslide from the incubator to the microscope
incubation chamber.
3. Several embryos can be imaged in a single experiment. As they
grow and are not physically attached, it is not practical to use
the automatic positioning program. Rather, position in X, Y,
and determination of optimal Z-stack have to be set up for each
embryo at each time-point. Before starting the time-lapse, one
should ensure optimal positioning of the embryos (Fig. 3). At
that stage of development, anterior-posterior axis can be determined through morphology alone. To visualize gastrulation
EMT, they should lie either on their posterior or lateral side,
which are natural positions due to the ellipsoid embryo shape.
Embryos can be gently flipped in the conical well using coarse
forceps in a closed position, or a plastic tip.
4. It is essential to minimize embryo exposure to light (see Note 6).
3.4 Image Analysis
Imaging data should be stored on two separate servers as backup.
Images are viewed and analyzed using an image analysis program
installed on a powerful workstation. Confocal and two photon
generated images are 3D reconstructed, projected on the Z axis,
and their brightness and contrast enhanced (Fig. 4). For time lapses
acquisitions, embryos must be registered using a drift correction
tool. Embryos are highlighted on each Z slice to generate a 3D
virtual embryo. The same embryo is registered across time by
matching its center of gravity. Individual cells are then segmented
through manually highlighting cell membranes along the Z slices
using a graphic tablet. The cells are then tracked over time using the
imaging program tracking algorithm. Tracking and cell shape data
can then be funneled through a dedicated python script [14] for
statistical analysis.
Ex vivo Imaging of EMT in the Early Mouse Embryo
141