Multiphoton microscopy for label-free multicolor imaging of peripheral nerve

Conventional histomorphometry of peripheral nerve entails lengthy chemical processing, ultrathin sectioning in resin, and imaging by light or electron microscopy. Multiphoton microscopy techniques exist enabling label-free and in vivo imaging of histological samples. Third-harmonic-generation microscopy has recently been demonstrated effective for imaging the myelin sheath of peripheral nerve axons in animal models. Herein, we characterize use of second and third harmonic generation microscopy for label-free imaging of murine and human peripheral nerve via a novel multicolor multiphoton microscope based on a single excitation wavelength at 1300 nm. Second harmonic generation signal from collagen centered about 650 nm delineates neural connective tissue, while third harmonic general signal centered about 433 nm delineates myelin and other lipids. In transgenic mice expressing yellow fluorescent protein linked to the thy1 promoter, three-photon-excitation with emission peak at 527 nm delineates axoplasm. We compare label-free multiphoton imaging of murine and human peripheral nerve against conventional chemical stains and discuss clinical implications of this approach in guiding intraoperative decision making in nerve transfer procedures.