American Society for Peripheral Nerve

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Novel techniques for high-throughput peripheral nerve histomorphometry
Iván Coto Hernández, PhD1, Wenlong Yang, PhD2, Lars Rishøj, PhD3, Suresh Mohan, MD4, Siddharth Ramachandran, PhD3 and Nate Jowett, MD5, (1)Mass Eye and Ear, Boston, MA, (2)Harvard University, Cambridge, MA, (3)Boston university, Boston, MA, (4)Mass. Eye and Ear/ Harvard Medical School, Boston, MA, (5)Facial Nerve Center - Dept. of Otolaryngology, Harvard Medical School / Massachusetts Eye and Ear, Boston, MA


Conventional nerve histomorphometry is resource intensive, necessitating complex sample preparation and error-prone axon quantification. Stimulated Raman scattering (SRS) microscopy and third harmonic generation (THG) are label-free, nonlinear optical processes with minimal sample preparation requirements that enable high-contrast and high-resolution imaging of histologic samples. Herein, we demonstrate the utility of these emerging imaging techniques for label-free resolution of myelin in rodent and human peripheral nerve. We further train a machine learning algorithm for rapid quantification of myelinated axons from imaged sections and demonstrate its applicability to surgical practice.


Fresh frozen sections of healthy rat sciatic nerve and human motor branch of obturator nerve were employed. Sections were left unstained and imaged by SRS and THG (using a custom-assembled multiphoton microscope), or stained with FluoroMyelin® and imaged by widefield fluorescent microscopy. Myelinated axon counts were automatically quantified across entire cross-sections using a trainable random forest machine learning algorithm.


Robust visualization of myelinated axons in unstained sections was possible with SRS and THG imaging. Rapid quantification of myelinated axon counts in healthy rodent and human nerve was achieved using a machine learning algorithm.


A rapid protocol for quantification of myelinated axon counts from peripheral nerves employing label-free imaging techniques with minimal sample preparation has been described.

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