layers) as well as that of their progeny when division occurs. A
similar approach could be used to record the dynamics of cell
organelles or specific proteins during EMT. The portfolio of conditional fluorescent reporter mouse lines is indeed constantly
increasing [13].
2 Materials
2.1 Large Equipment
1. Stereomicroscope with transmitted light and magnification
ranging from approximately 10Â to 40Â for embryo
dissection.
2. Humidified CO 2 incubator.
3. Laser scanning confocal or 2-photon inverted microscope
(Fig. 1) with 5Â, 10Â, 20 or 25Â, and 40Â objectives (for
example, a LD LCI Plan Apochromat 25Â/0.8 Imm Korr DIC
M27 (WD 570 um) objective or LD C Apochromat 40Â/
1.1 W Korr M27 (WD 620 μm) for detailed acquisitions). 5Â
and 10Â dry objectives are used to identify and position samples, and 20 or 25Â immersion objectives are used to record a
global view of the embryo. To visualize individual cells, 40Â
immersion objective is usually most appropriate. The microscope must be placed on an anti-vibration air table. The
on-stage environmental culture chamber allows temperature
control (37
C), proper humidification, and gas control
(CO 2 /O 2 /N 2 ) (see Note 1).
4. Computer workstation with sufficient RAM, adequate graphic
card for the image data acquisition and processing software,
and consequent storage capacity (as an example, we use a
computer equipped with Dual Intel Xeon Processor (Eight
Core HT, 3.4 GHz Turbo, 25 MB), 64 GB 1866MHz
DDR3 ECC RDIMM RAM, NVIDIA 980 GTX graphics
card, and running Windows 7, 64 bit). Acquisition software
available with commercial microscopes (such as Zeiss’ ZEN,
Leica’s LASX or MetaMorph) and open-source software such
as ImageJ or Fiji allow basic image analysis. Commercially
available software packages provide additional features for
3D/4D analysis and representation. However, there is no
generic software for detailed analysis of cell contours that will
be efficient in any 3D context. Depending on the volume of
data, one can either develop dedicated software or rely on
manual segmentation, which is greatly facilitated by the use of
a graphic tablet (such as Wacom CINTIQ 13HD or
equivalent).
Ex vivo Imaging of EMT in the Early Mouse Embryo
137
similar approach could be used to record the dynamics of cell
organelles or specific proteins during EMT. The portfolio of conditional fluorescent reporter mouse lines is indeed constantly
increasing [13].
2 Materials
2.1 Large Equipment
1. Stereomicroscope with transmitted light and magnification
ranging from approximately 10Â to 40Â for embryo
dissection.
2. Humidified CO 2 incubator.
3. Laser scanning confocal or 2-photon inverted microscope
(Fig. 1) with 5Â, 10Â, 20 or 25Â, and 40Â objectives (for
example, a LD LCI Plan Apochromat 25Â/0.8 Imm Korr DIC
M27 (WD 570 um) objective or LD C Apochromat 40Â/
1.1 W Korr M27 (WD 620 μm) for detailed acquisitions). 5Â
and 10Â dry objectives are used to identify and position samples, and 20 or 25Â immersion objectives are used to record a
global view of the embryo. To visualize individual cells, 40Â
immersion objective is usually most appropriate. The microscope must be placed on an anti-vibration air table. The
on-stage environmental culture chamber allows temperature
control (37
C), proper humidification, and gas control
(CO 2 /O 2 /N 2 ) (see Note 1).
4. Computer workstation with sufficient RAM, adequate graphic
card for the image data acquisition and processing software,
and consequent storage capacity (as an example, we use a
computer equipped with Dual Intel Xeon Processor (Eight
Core HT, 3.4 GHz Turbo, 25 MB), 64 GB 1866MHz
DDR3 ECC RDIMM RAM, NVIDIA 980 GTX graphics
card, and running Windows 7, 64 bit). Acquisition software
available with commercial microscopes (such as Zeiss’ ZEN,
Leica’s LASX or MetaMorph) and open-source software such
as ImageJ or Fiji allow basic image analysis. Commercially
available software packages provide additional features for
3D/4D analysis and representation. However, there is no
generic software for detailed analysis of cell contours that will
be efficient in any 3D context. Depending on the volume of
data, one can either develop dedicated software or rely on
manual segmentation, which is greatly facilitated by the use of
a graphic tablet (such as Wacom CINTIQ 13HD or
equivalent).
Ex vivo Imaging of EMT in the Early Mouse Embryo
137