For each age condition, the analysis of PSHG signals was performed on images taken in different regions of the whole sample surface. Only pixels exhibiting a total SHG signal above are considered, which ensures that the measurements are not dominated by noise.16 Typical retrieved images for the orientation () and molecular order () parameters are shown in Fig. 3(a). is found to be close to 0 in all cases, indicating cylindrical-symmetry distributions along the fibrils.16 The angle is well correlated with the macroscopic fibril orientation. The -image shows a large distribution of values, most probably due to intrinsic heterogeneities in fibril morphologies and to the diversity present within the rat’s population of each age-group. However, in spite of such intra-age heterogeneities, inter-age variations are pronounced and can be clearly observed [Fig. 3(c), ANOVA on subregions], with a significance probability below 5% except between adult and young adult cases. The maximum molecular order (high values) is obtained for the newborn and old collagens. Since molecular order is representative of how fibrils are oriented with respect to each other, a large value indicates a low degree of orientational freedom for molecules constituting the fibrils, also interpreted as a lower aperture of their angular distribution [Fig. 3(d)].9–11,16 The large value observed in the newborn group can therefore be attributed to higher structural constraint and rigidity, which is a priori contradictory with the higher elastic modulus observed in newborn collagen three-dimensional (3-D) matrices.17 Elasticity can be however ascribed to a more macroscopic scale effect related to the length of fibrils, which appears to increase with age. Finally, young adult fibrils show a lower molecular order, signature of a more complex assembly of individual fibrils within their thicker fibers.