Initial experience with label-free stimulated Raman scattering microscopy for intraoperative assessment of peripheral nerves.

Background: During nerve repair, an intraoperative assessment of the quality of the nerve stump is critically important for achieving a good outcome. Frozen section analysis of osmium-hematoxylin stained sections has not been adopted at many centers, including ours. This has left us with bread-loafing the nerve and visually assessing for healthy fascicles. A technique that would allow for rapid, safe, quantitative intraoperative assessment of nerve quality, including myelin quantity, would be beneficial. Stimulated Raman Scattering (SRS) microscopy is a rapid, label-free technique that images lipids well that may be uniquely suited for this purpose.

Objective: To describe our initial experience and lessons learned using SRS microscopy for evaluation of peripheral nerve tissue.

Methods: We present 6 cases during which SRS microscopy was used to evaluate peripheral nerve tissue, including standard histology and SRS microscopy images, where applicable.

Results: Our current technique involves OCT embedding the nerve tissue and then cutting 70 µm sections on a standard cryostat. The SRS microscope slide is modified to change the buffer depth from 100 µm to 50 µm. We analyzed the gray scale composite images, merged from the CH (lipid) channel and CH (protein) channel. This technique reliably produced cross-sectional images and showed good capability for imaging myelinated axons within fascicles.

Conclusions: We demonstrate here an innovative approach to quantifying myelin in peripheral nerve using Stimulated Raman Scattering microscopy. This should prove useful in the care and surgical treatment of patients with peripheral nerve injuries.