Image analysis was performed as follows. In IMAGE J (Rasband, W. S., IMAGE J, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2011), the background in each channel (SHG and IF) was defined by the average pixel counts of an image with no excitation laser and subtracted from the raw SHG and IF images, respectively. A single common threshold was then chosen by a blinded observer which passed the most collagen pixels while rejecting the most background pixels (typically individual pixels not clearly laying in a fibrillar pattern). The common threshold was then applied to all SHG images, setting collagen fiber pixels to 1 and any remaining dim background pixels to zero, producing an “SHG mask.” The same thresholding procedure was followed with IF images, producing an “IF mask.” The SHG image was multiplied by the SHG mask, producing the “masked SHG image,” and the IF image was multiplied by the IF mask to produce the “masked IF image.” The average pixel count of the masked SHG image, divided by the average pixel count of the SHG mask, is then the average pixel count of those pixels above threshold, i.e., within collagen fibers, likewise with the masked IF image. The average SHG pixel count, average IF pixel count, and their ratio, of those pixels within collagen fibers, are then reported as the images’ SHG, IF, and OI, respectively. Note that due to fluctuations in pixel counts, there are occasional pixels which are above threshold in the SHG image but below threshold (and hence set to zero) in the IF image. Consequently, the SHG/IF ratio is infinity for those pixels. This makes it problematic to calculate an average SHG/IF for the imaged region by first producing an SHG/IF ratio image and then calculating the average pixel count of that image. We avoid this problem by first separately calculating the average SHG and IF pixel counts of the whole imaged region and then dividing these two (nonzero) numbers to produce the SHG/IF for the whole imaged region.