Multimodal Fourier transform infrared spectroscopy and second- and third-harmonic generation imaging for integrated...

Fourier Transform Infrared (FTIR) spectroscopy combined with nonlinear optical imaging was employed to characterize molecular and structural alterations associated with oral squamous cell carcinoma (OSCC) progression in control, margin, and tumor tissues. Spectral and imaging datasets were evaluated using multivariate statistical analysis and receiver operating characteristic (ROC) analysis to identify disease-associated features and assess their discriminatory performance. Integration of second- and third-harmonic generation (SHG/THG) imaging with histopathological staining provided complementary information on tissue architecture, with SHG contrast reflecting extracellular matrix and collagen organization and THG contrast highlighting cellular and nuclear features. FTIR analysis revealed coordinated biochemical alterations during OSCC progression, including reduced α-helical protein content with preservation of β-sheet features, lipid remodeling characterized by decreased ester carbonyl content, lower CH₂/CH₃ ratios, and reduced lipid unsaturation, altered glycogen-related signals, and nucleic acid reorganization characterized by decreased DNA and increased RNA contributions. Although many spectral parameters showed statistically significant differences, their individual diagnostic performance was limited, particularly by low specificity. A selected set of spectral parameters, including the Amide I / Ester carbonyl ratio, α-helix content, α-helix/β-sheet ratio, and nucleic acid-associated markers, showed relatively higher discriminatory performance, with AUC values of 0.80-0.93, sensitivities of 66-93%, and specificities of 1-28%. These findings demonstrate that FTIR spectroscopy captures distinct molecular alterations associated with OSCC progression, while SHG/THG imaging provides complementary structural information. Their integration therefore offers a multimodal, label-free framework for spectro-histopathological characterization of OSCC and supports the use of multiple biochemical and structural descriptors rather than individual spectral markers.