Fig. 7. Multiphoton microscope imaging of developing chicken skin explant cultures.

A) The embryonic E6 skin is labeled with Hoechst 33342 dye and cultured as an explant. Serial images from the surface to the bottom are reconstituted into a three-dimensional image. The nuclei at different depths from the surface are graded with pseudocolor from blue (epithelial surface) to red (50 µm from surface) to facilitate single cell tracing. The left picture shows the three-dimensional distributions of cells and the right panel shows a bottom view of the nuclei in an en face projection to X-Y plane. The X or Y axis is 140 mm and the Z axis is 50 mm.

B) Time-lapse multiphoton tracing of cell rearrangement. To highlight the mesenchymal cell movement, the dermal side is on the top and epidermal side at the bottom. The depth-graded pseudocolor helps to delineate individual cells and facilitate single cell tracing. There is a trend of movement toward the right hand side of the figure and to the dermal side. The X or Y axis is 140 mm and the Z axis is 50 mm.

C) Multiphoton auto-fluorescence and second harmonic generation (SHG) images of unstained developing feather bud. The upper panel shows the images of E6 skin at different depths before dermal condensation formation. In E6, the dermal cells have an even cell distribution and there is scanty SHG signals from collagen (−88 and −124 mm). The epithelial cells can also be visualized with an autofluorescent cytoplasm and a nuclear halo (−8 and −20 mm).The lower panel shows the images of E7 skin at different depths when dermal condensates appear. In the lower power view of E7 skin, single cells cannot be delineated. The dermal condensates have higher autofluorescence due to the higher cell density and interbud area is rich in SHG signals from the collagen. The low power image clearly demonstrates the preferential cell and collagen distribution in the developing skin. Autofluorescence is green and second harmonic generation is red; bars: 100 mm.