Multiphoton microscopy for label-free multicolor imaging of peripheral nerve

Area of Science:

• Neuroscience
• Biomedical Optics
• Histology

Background:

• Quantifying myelinated fibers in peripheral nerves is crucial for diagnosing and managing nerve disorders.
• Label-free in vivo imaging of peripheral nerves can be achieved using multiphoton microscopy techniques, such as third-harmonic generation.

Purpose of the Study:

• To develop a multiphoton microscope utilizing a custom high-power infrared fiber laser for label-free peripheral nerve imaging.
• To enable stain-free multicolor imaging of both murine and human peripheral nerve tissue.

Main Methods:

• Designed and implemented a cost-effective multiphoton microscope system.
• Employed a single fiber laser source operating at 1300 nm.
• Utilized second-harmonic generation (SHG) for collagen imaging and third-harmonic generation (THG) for myelin and lipid imaging.

Main Results:

• SHG signals at ~650 nm delineated neural connective tissue, while THG signals at ~433 nm identified myelin and lipids.
• Three-photon excitation with emission at 527 nm visualized axoplasm in transgenic mice.
• Adequate signal was obtained from unlabeled samples for myelinated fiber segmentation, achieving imaging depths over 100 μm.

Conclusions:

• A fiber laser-powered multiphoton microscope facilitates stain-free histomorphometry of mammalian peripheral nerve.
• The microscope's simplified design offers potential for clinical translation in diagnosing peripheral nerve disorders.