Multiphoton microscopy for label-free multicolor imaging of peripheral nerve

Lars Rishøj, Ivan Coto Hernandez, Siddharth Ramachandran, Nate Jowett

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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 100-mu m 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. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License.
Original languageEnglish
Article number056501
JournalJournal of Biomedical Optics
Issue number5
Number of pages11
Publication statusPublished - 2022


  • Lasers
  • Imaging
  • Label-free
  • Nonlinear optical microscopy
  • Nerve fibers
  • Myelinated


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