Figure 9

SHG-B and SHG-F identify different collagen fibers. A Z-stack of images 78 μm deep was collected of a TEB from a Carmine Alum whole mount. A. A two-color image of SHG-B and SHG-F signals illustrates that even in one XY plane, two types of collagen fibers are identified. B. An orthogonal view of the same region showing SHG-B and SHG-F signals at various depths. Three layers of fibers are seen with the TEB which is sandwiched between two of the layers, asterisk. Dashed lines indicate planes of associated images; red for YZ, green for XZ, and blue for XY. C and D. SHG-B (red) / 500–550 nm (autofluorescence, blue)/ SHG-F (green) images reveal that SHG-B and SHG-F signals are not identical despite their arrangement in parallel arrays. In D, Individual fibers can be seen to have both SHG-B and SHG-F signals. Typically the signal from SHG-B (red) has more texture, whereas the signal from SHG-F (green) is smoother and finer in appearance. A-B, E-G. Small vessels contain associated fibers with both SHG-F and SHG-B showing strong signal intensity compared with with TEB-associated fibers that are indicated by arrows. F-G are insets from E indicated in E by dashed boxes. E scale bar = 50 μm; C, F-H, scale bar = 20 μm; D, scale bar = 10 μm.