Figure 3

Comparison of reflected (SHG-B) and transmitted SHG-F signals reporting surface collagen layers. At Ex 860 nm, SHG-B (ChS: 393–436 nm; red) and SHG-F (ChD, green) images were collected in a Z-series beginning at the lateral margin of the gland. A-B. The patterns of SHG-B and SHG-F are not identical since single color fibers appear red (SHG-B) or green (SHG-F) as shown in XY planes (A) and in orthogonal images (XY; XZ; YZ) (B). C. 3D images were prepared using Metamorph Offline Ver. 7.7.7.1 (“Open in 4D viewer”). The surface shown rested adjacent to the coverslip surface. The SHG-F view bears the pattern of the outlines of fat cells, whereas the view of fibers provided by SHG-B is not affected. D-E. A single XY slice reveals details of the fiber orientation and was taken at a depth of 4 μm. The average intensities of SHG-B signals for three different ROIs were calculated and plotted against Z-depth on the X-axis. The thickness of the fibrillar layer is on the order of 6 μm, although SHG is detected variably deeper into the tissue. Scale bars = 50 μm.