Label-Free Identification of Tumor Tissues by Coherent Nonlinear Vibrational Mode Imaging

We present vibrational sum-frequency generation (VSFG) microscopy as a new label-free chemical imaging technique for tumor identification. This method combines the chemical-bond selectivity of vibrational spectroscopy with coherent interference of second-order coherent nonlinear optics. Using a fast line-scanning VSFG microscope, we obtained hyperspectral VSFG images of collagen I from both lung tissues bearing metastatic tumors and in tumor-free ones, which reveal drastic different spectral signatures: tumor samples exhibit large NH stretch (NHS) and CH stretch (CHS) versus the CH2 symmetric stretch (CH2,Ss), compared to healthy controls. We then identified two key spectral signatures to distinguish metastatic tumor and tumor-free tissues: the intensity ratio of NHS/CH2,Ss and CHS/CH2,Ss modes. These signatures demonstrated high fidelity in distinguishing between tumors and normal tissue in both mouse and human lung samples. Theoretical modeling indicates that distinctive interferences of spectral peaks are sensitive to interfibrillar distances, at 130 of nanometer level. These findings suggest that collagen fibrils are more densely packed in tumors, corroborating the enhanced stiffness observed in tumor tissues. VSFG microscopy offers a highly selective, label-free chemical imaging characterization that preserves sample integrity, making it a valuable tool for oncology, pathology and fundamental biophysical research.