2.3 Image
Processing
1. Computer workstation with 8 Gb RAM and capability to run
MatLab and ImageJ.
2. Between 1 and 2 Tb storage for archiving raw, denoised, and
analyzed data (see Note 5).
3 Methods
3.1 Tissue
Dissection, Culture,
and Mounting
1. Incubate fertile eggs (see Note 6), transgenic for memGFP at
38
C in a humidified incubator until HH14/15 or desired
stage (see Note 7).
2. Harvest embryos (see Note 8) in culture medium, pre-warmed
to 38
C, in petri dishes coated with silicone lining the base of
the dishes (see Note 9).
3. Pin down embryos on the silicone dish using insect pins,
stretching the extraembryonic tissue (see Note 10).
4. Use fine scalpels to dissect the explants containing five to six
somite pairs; for imaging of the most posterior epithelial
somites, ensure that the pre-segmented mesoderm is included.
Ensure that the somites are flanked by intermediate and lateral
plate mesoderm, with surface ectoderm above and endoderm
below, as well as notochord and neural tube in the axial midline
(see Note 11).
5. Prepare low melting point agarose in culture medium (2% w/v)
(no phenol red) with 10% serum and 1% penicillin/
streptomycin.
6. Place a small amount (200 μL) of the agarose/medium on the
base of the glass slide, on the glass-bottom dishes.
7. While setting, transfer the dissected tissue (see Note 12) and
position it with the dorsal side toward the base (see Note 13),
so that the imaging starts from dorsal through to the ventral
side on an inverted microscope as shown in Fig. 1.
8. Add warm agarose/medium on the somite explants until it is
fully covered.
9. Place the dishes on ice for 5 min to fully set.
10. Then place inside a humidified and heated chamber (38
C) for
microscopy (see Notes 14 and 15).
3.2 Long-Term
Two-Photon
Microscopy
1. Acquire images at 10Â magnification to view a string of 3–4
somites (depending on stage/size) or 20Â magnification to
view an individual somite, starting at the interface and imaging
up through the tissue at 510 nm Z slices. Return to the interface and repeat every 20 min. Record images as Tiff files (see
Notes 16 and 17).
4D Live Imaging and Analysis of Chick Embryo Somites
177
Processing
1. Computer workstation with 8 Gb RAM and capability to run
MatLab and ImageJ.
2. Between 1 and 2 Tb storage for archiving raw, denoised, and
analyzed data (see Note 5).
3 Methods
3.1 Tissue
Dissection, Culture,
and Mounting
1. Incubate fertile eggs (see Note 6), transgenic for memGFP at
38
C in a humidified incubator until HH14/15 or desired
stage (see Note 7).
2. Harvest embryos (see Note 8) in culture medium, pre-warmed
to 38
C, in petri dishes coated with silicone lining the base of
the dishes (see Note 9).
3. Pin down embryos on the silicone dish using insect pins,
stretching the extraembryonic tissue (see Note 10).
4. Use fine scalpels to dissect the explants containing five to six
somite pairs; for imaging of the most posterior epithelial
somites, ensure that the pre-segmented mesoderm is included.
Ensure that the somites are flanked by intermediate and lateral
plate mesoderm, with surface ectoderm above and endoderm
below, as well as notochord and neural tube in the axial midline
(see Note 11).
5. Prepare low melting point agarose in culture medium (2% w/v)
(no phenol red) with 10% serum and 1% penicillin/
streptomycin.
6. Place a small amount (200 μL) of the agarose/medium on the
base of the glass slide, on the glass-bottom dishes.
7. While setting, transfer the dissected tissue (see Note 12) and
position it with the dorsal side toward the base (see Note 13),
so that the imaging starts from dorsal through to the ventral
side on an inverted microscope as shown in Fig. 1.
8. Add warm agarose/medium on the somite explants until it is
fully covered.
9. Place the dishes on ice for 5 min to fully set.
10. Then place inside a humidified and heated chamber (38
C) for
microscopy (see Notes 14 and 15).
3.2 Long-Term
Two-Photon
Microscopy
1. Acquire images at 10Â magnification to view a string of 3–4
somites (depending on stage/size) or 20Â magnification to
view an individual somite, starting at the interface and imaging
up through the tissue at 510 nm Z slices. Return to the interface and repeat every 20 min. Record images as Tiff files (see
Notes 16 and 17).
4D Live Imaging and Analysis of Chick Embryo Somites
177