Pathological investigation on healthy ovarian tissue sections by an integrated label-free multimodality optical microscopy

Nonlinear multimodal imaging integrates nonlinear optical techniques with multimodal imaging approaches, providing a powerful research tool for investigating the tumor microenvironment and pathological mechanisms of malignancy through its label-free operation and high-resolution capabilities. We have developed an integrated multimodal system combining nonlinear optical imaging (NLOI) and confocal Raman microscopic imaging (CRMI) to achieve comprehensive, label-free multi-dimensional characterization of both tissue architecture and biochemical composition in ovarian specimens. The system architecture features an erbium-doped fiber laser-based NLOI module for simultaneous three-photon fluorescence (3PEF), second-harmonic generation (SHG) and third-harmonic generation (THG) imaging, coupled with an 830[Formula: see text]nm continuous-wave laser for molecular fingerprint analysis via a CRMI module. Experimental results demonstrated that NLOI clearly revealed the distribution of collagen fibers (SHG) in ovarian vascular walls, cellular interface structures (THG) and metabolic activity characteristics (3PEF), while phosphatidic acid and hydroxyproline are predominantly localized within the vascular adventitia; phenylalanine enrichment predominantly in the intima and media layers; cholesterol esters were distributed throughout the vascular wall; collagen concentration gradients across the media and adventitia, and distinctive distribution patterns of nucleic acids and lipids within endothelial cells and smooth muscle cells. Most notably, quantitative texture analysis through FFT and GLCM algorithms revealed a periodic arrangement of collagen fibers in healthy ovarian vasculature, providing novel experimental evidence for understanding vascular wall biomechanical stability. This multimodal system significantly enhances the precision and depth of label-free tissue analysis, demonstrating important application potential in biological research and clinical diagnosis of diseases such as ovarian cancer.