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Background:. Clinical outcomes after nerve injury and repair remain suboptimal. Patients may be plagued by poor functional recovery and painful neuroma at the repair site, characterized by disorganized collagen and sprouting axons. Collagen deposition during wound healing can be intrinsically imaged using second harmonic generation (SHG) microscopy. The purpose of this study was to develop a protocol for SHG imaging of nerves and to assess whether collagen alignment can be quantified after nerve repair. Methods:. Sciatic nerve transection and epineural repair was performed in male rats. The contralateral nerves were used as intra-animal controls. Ten-millimeter nerve segments were harvested and fixed onto slides. SHG images were collected using a 20× objective on a multiphoton microscope. Collagen fiber alignment was calculated using CurveAlign software. Alignment was calculated on a scale from 0 to 1, where 1 represents perfect alignment. Statistical analysis was performed using a linear mixed-effects model. Results:. Eight male rats underwent right sciatic nerve repair using 9-0 Nylon suture. There were gross variations in collagen fiber organization in the repaired nerves compared with the controls. Quantitatively, collagen fibers were more aligned in the control nerves (mean alignment 0.754, SE 0.055) than in the repairs (mean alignment 0.413, SE 0.047; P < 0.001). Conclusions:. SHG microscopy can be used to quantitate collagen after nerve repair via fiber alignment. Given that the development of neuroma likely reflects aberrant wound healing, ex vivo and/or in vivo SHG imaging may be useful for further investigation of the variables predisposing to neuroma. more...