Stainless Steel vs Calcium Fluoride for biomedical Raman Spectroscopy

Raman spectroscopy (RS) has been shown to differentiate various disease states in tissue samples, yet the impact of sample substrate on spectral quality and downstream machine-learning (ML) performance is under-explored. We collected 960 spectra from 36 formalin-fixed, paraffin-embedded colonic specimens mounted in parallel on calcium fluoride ( CaF ₂ ) and mirrored stainless-steel ( SS ) slides. Raw SS spectra were more intense than CaF ₂ , and routine dewaxing left prominent paraffin peaks on CaF ₂ that were largely absent on SS . Extending CaF ₂ xylene immersion to 48 h reduced key paraffin peaks by 11–23 %, aligning its biochemical profile with SS . Nested cross-validated classifiers were built to distinguish true disease (T1) from epithelial misplacement (EM). The highest accuracies were observed on SS (0.65–0.66), while CaF ₂ improved from 0.41–0.57 to 0.65–0.69 after extended dewaxing, chiefly via gains in recall. Although optimal dewaxing renders CaF ₂ diagnostically non-inferior, SS still offers superior accuracy, faster preparation, at approximately 4% of the cost, supporting its integration into scalable, AI-enabled RS pathology workflows.