Figure 2

Multiphoton microscopy of live GFP-mouse mammary gland, ex vivo. In each series (A-C and D), imaging acquisition began at the margin of the mammary gland resting on the coverslip and extended to depths of 90 μm (A) and 72 μm (D) including a terminal end bud (TEB). The margin of the mammary gland is identified by the dense collagen/ ECM layer visualized by SHG-B (red) that is absent in the gland interior. The TEB volume (GFP images, green) excludes the SHG-B signal. (B) is average intensity plotted on the Y-axis and Z-image depth plotted on the X-axis. Arrows indicate image depths shown in A (6 μm, 15 μm, 25 μm, and 48 μm). The boxed regions of the 3D reconstruction image (A, 3D) includes the position of R01’s used for quantification of SHG-B and GFP signal intensities in B (ROIs are indicated by white boxes “TEB” and “non-TEB”). In C, the same TEB was imaged with a Zeiss c-Apochromat 40X/ 1.2 NA W corr. lens. Although this lens has a shorter working distance, cellular details can be obtained at higher resolution when the TEB is relatively close to the surface of the mammary gland. At Z = 35 μm, the body cells of a TEB (“Body cells”) appear very bright and separated by non-GFP containing spaces. At Z = 56 μm, a position midway into the TEB in the Z-dimension, cap cells at the tip of the TEB are seen as a continuous layer with an outer, smooth margin (arrows, “Cap”). D. A dense array of cells (GFP) is poorly imaged since the adipose tissue scatters light and obscures GFP in a pattern reflecting the shape of the fat cells (arrow, z = 46 μm). Scale bars A, D = 50 μm; in C, 20 μm.