Differentiation of Healthy Ex Vivo Bovine Tissues Using Raman Spectroscopy and Interpretable Machine Learning

Objectives: Integrating machine learning with Raman spectroscopy (RS) shows strong potential for intraoperative guidance in orthopedic procedures, but limited algorithm transparency remains a barrier to clinician trust. This study aims to develop interpretable machine learning models capable of accurately classifying bovine tissue types (bone, bone marrow, fat, and muscle) relevant to orthopedic surgery by identifying key Raman biomarkers to improve model transparency. Methods: A portable RS system equipped with a 785 nm fiber-optic probe was used to collect spectral data from excised bovine tissues, including bone, bone marrow, muscle, and fat. One-dimensional convolutional neural network (1D-CNN) and support vector machine (SVM) models were developed to classify these tissue types. The Raman spectral data were divided using a sample-based, stratified splitting strategy and evaluated across 30 independent iterations. Feature importance maps were generated for both models, and matching scores were calculated to correlate significant spectral features with known Raman biomarkers. Results: Through feature importance analysis and matching scores generated by the 1D-CNN and SVM models, critical Raman biomarkers—including hydroxyapatite, lipids, amino acids, and collagen—were identified as essential for distinguishing between the different bovine tissue types, providing deeper insights into their molecular differences. Conclusions: The integration of interpretable machine learning models with RS enabled accurate differentiation of bovine tissues relevant to orthopedic surgery, while enhancing model transparency through biomarker identification. Linking model predictions to biologically meaningful Raman features supports the development of RS as a reliable tool for precision-guided surgical procedures.