Experimental workflow—Collagen remodeling analysis using image spatial correlation. Upper right panel: collagen SHG images were acquired by 2-photon microscopy. 10 -stack slices across 200 μm depth from culture dish surface were collected. Each slice covers 850 μm × 850 μm. Upper left panel: an example of collagen SHG images from close to the culture dish surface (slice 1) to 200 μm above (slice 10). The SHG intensity decreases with the increasing depth of acquisition. However, since image spatial correlation is not sensitive to average intensity change, image spatial correlation (1) was applied to each slice, and the results were averaged to produce one correlation image. Lower left panel: the spatial correlation image calculated from the SHG -stack. The relatively symmetrical autocorrelation pattern indicates that with our sample preparation, collagen fibers were not preferentially aligned toward specific direction at the scale of the whole image. Lower right panel: the and cross sections from the spatial correlation image were fitted with two Gaussian components. One component has the standard deviation close to pixel size (green curve); the other component has broader standard deviation (blue curve), and its width varies based on the collagen compaction level.