Frozen agarose–assisted tissue preparation for stimulated Raman histology enables rapid intraoperative assessment of peripheral nerve integrity

Background Intraoperative assessment of peripheral nerve viability during traumatic nerve repair is predominantly based on the surgeon’s subjective macroscopic criteria. Frozen section histology can provide objective information, its routine, repeated use is however limited by processing time. Intraoperative Stimulated Raman Histology (SRH) enables rapid, label-free imaging of native tissue. Current intraoperative SRH workflows relying on squash preparations that do not adequately preserve peripheral nerve microarchitecture. Objective To establish a rapid tissue preparation protocol that preserves true cross-sectional nerve architecture and investigate the feasibility of SRH for the assessment of peripheral nerve resection margins. Methods Peripheral nerve tissue from 15 patients was analyzed using SRH. Various tissue preparation strategies were systematically assessed, including freehand and guided sectioning of native, fixed and agarose embedded short-term frozen peripheral nerve samples. Workflow duration and image quality were evaluated descriptively. Results Freehand sectioning resulted in substantial architectural distortion, preventing reliable evaluation of fascicular organization. Guided sectioning improved thickness control but remained limited by tissue deformation during the cutting process. Agarose embedding followed by one-minute dry ice exposure enabled reproducible generation of 500 μm cross-sections with well-preserved fascicular architecture and minimal artifacts in SRH. Tissue preparation required 3.5 min. Conclusion Frozen-agarose assisted tissue preparation for SRH (FA-SRH) is feasible and compatible with surgical workflows, providing rapid visualization of peripheral nerve microarchitecture. Therefore, FA-SRH represents a promising complementary tool to support objective intraoperative decision-making during peripheral nerve repair surgery.