9. Fertile transgenic eggs; embryos ubiquitously expressing a
membrane-bound GFP (memGFP), obtained from Roslin
Institute, University of Edinburgh, Scotland, UK.
10. Incubator (38
C).
11. Silicone elastomer (Sylgard 184 silicone, Dow Corning, USA
or equivalent).
12. Non-coated 35 mm glass-bottom dishes with 7 mm glass
diameter.
2.2 Time-Lapse
Video-Microscopy
1. Inverted two-photon microscope equipped with 10 and 20Â
objectives and a motorized stage for multiple XYZ acquisition
(see Note 1).
2. Environmental chamber to control temperature (38
C) and
CO 2 (5%) (see Notes 2 and 3).
3. A software with time-lapse and multiple XYZ positions modules to control the microscope (see Note 4).
Fig. 3 Examples of cellular dynamics and behaviors that can be observed. (a) The panels show a series of
images in the same optical plane from t ¼ 0 to t ¼ 140 min. Cells that round up and then divide can be seen
and are indicated by white arrow heads, scale bar ¼ 10 μm. (b) The top panel (1) shows an optical section
with somite cells extending filopodia toward the overlying ectoderm, (2) is a deeper optical section where cells
extending filopodia toward the intermediate mesoderm laterally can be observed. (c) The panels show a series
of images focusing on the medial lip of the somite, adjacent to the neural tube visible in the bottom corner on
the left. The red tracing indicates a cell that is detaching from the lip and moves into the somite, where it most
likely contributes to the emerging myotome (t ¼ min)
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Gi Fay Mok et al.
membrane-bound GFP (memGFP), obtained from Roslin
Institute, University of Edinburgh, Scotland, UK.
10. Incubator (38
C).
11. Silicone elastomer (Sylgard 184 silicone, Dow Corning, USA
or equivalent).
12. Non-coated 35 mm glass-bottom dishes with 7 mm glass
diameter.
2.2 Time-Lapse
Video-Microscopy
1. Inverted two-photon microscope equipped with 10 and 20Â
objectives and a motorized stage for multiple XYZ acquisition
(see Note 1).
2. Environmental chamber to control temperature (38
C) and
CO 2 (5%) (see Notes 2 and 3).
3. A software with time-lapse and multiple XYZ positions modules to control the microscope (see Note 4).
Fig. 3 Examples of cellular dynamics and behaviors that can be observed. (a) The panels show a series of
images in the same optical plane from t ¼ 0 to t ¼ 140 min. Cells that round up and then divide can be seen
and are indicated by white arrow heads, scale bar ¼ 10 μm. (b) The top panel (1) shows an optical section
with somite cells extending filopodia toward the overlying ectoderm, (2) is a deeper optical section where cells
extending filopodia toward the intermediate mesoderm laterally can be observed. (c) The panels show a series
of images focusing on the medial lip of the somite, adjacent to the neural tube visible in the bottom corner on
the left. The red tracing indicates a cell that is detaching from the lip and moves into the somite, where it most
likely contributes to the emerging myotome (t ¼ min)
176
Gi Fay Mok et al.