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Aim: With the objective to investigate the mechanisms underlying absence of spontaneous axonal regeneration following spinal cord injury (SCI) we employ a multimodal label-free imaging approach to monitor glial scar in a mice SCI model. Method: To determine the relevant structural signature, and the nanobiomechanical behavior of healthy and injured spinal cord tissue we combine the non-linear, multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM). The glial scar at different post lesion time-points is investigated with these two techniques to monitor structural and elasticity (Young modulus) changes of the tissue.Results: 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues were recorded in MPM at 72h, 1week and 6 weeks post-lesion. The MPM images revealed the apparition of a strong SHG signal at 1week post injury, due to the formation of fibrillar collagen fibers (collagen type I) by the injury site in the glial scar. At 6 weeks’post-injury, the SHG signal is more intense and a higher number of fibers are detected in average. We further assessed the preferential orientation of the collagen bundles performing polarization dependent measurements of the SHG signal. The AFM based force spectroscopy measurements have been performed at the same post-lesion time-points to map the elastic properties of the healthy (grey and white matters) and injured (lesion) parts in the spinal cord tissue. The results suggested an increase of the lesion area stiffness over time that could be correlated with the apparition of fibrillar collagen observed in MPM, indicating the presence of a fibrotic process seven days after injury, that develops in time. As tissue stiffness is a regulator of neuronal growth, such kind of measurements might help to understand why adult mammalian axons do not regenerate after an injury. Our next step is to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage in mice that underwent SCI) on the structure and mechanical properties of the lesion site at 1 week and 6 weeks post injury.