Multiphoton microscopy for label-free multicolor imaging of peripheral nerve

SIGNIFICANCE: Means for quantitation of myelinated fibers in peripheral nerve may guide diagnosis and clinical decision making in management of peripheral nerve disorders. Multiphoton microscopy techniques such as the third-harmonic generation enable label-free in vivo imaging of peripheral nerves. AIM: Develop a multiphoton microscope based on a custom high-power infrared fiber laser for label-free imaging of peripheral nerve. APPROACH: A cost-effective multiphoton microscope employing a single fiber laser source at 1300 nm was designed and used for stain-free multicolor imaging of murine and human peripheral nerve. RESULTS: Second-harmonic generation signal from collagen centered about 650-nm delineated neural connective tissue, whereas third-harmonic general signal centered about 433-nm delineated myelin and other lipids. In sciatic nerve from transgenic reporter mice expressing yellow fluorescent protein within peripheral neurons, three-photon-excitation with emission peak at 527-nm delineated axoplasm. The signal obtained from unlabeled axially sectioned samples was adequate for segmentation of myelinated fibers using commercial image processing software. In unlabeled whole mount specimens, imaging depths over [Formula: see text] were achieved. CONCLUSIONS: A multiphoton microscope powered by a fiber laser enables stain-free histomorphometry of mammalian peripheral nerve. The simplicity of the microscope design carries potential for clinical translation to inform decision making in peripheral nerve disorders.