Figure 5. Control sample: By orientation distinguishable smooth muscle layers in the small intestine.

In order to verify our image processing method, we applied the exact same preparation and staining method to a rat small intestine, an organ with clearly delineated longitudinal and circumferential smooth muscle layers [8], [10]. Panels A–C show orientation distributions analogous to the panels in Figure 4 , at three different sites in the intestine. Panel D shows the average orientation distribution, calculated from panels A–C. All panels clearly show a transition from a superficial, longitudinal smooth muscle orientation to a circumferential orientation at the deep end. The patterns shown are all slightly shifted to the right by ∼10°, which is caused by the fact that the two pipettes used to mount the intestine were not perfectly aligned (i.e., microscopy images were rotated by ∼10°).