Nonlinear Imaging Detection of Organ Fibrosis in Minute Samples for Early Stage Utilizing Dual-Channel Two-Photon and Second-Harmonic Excitation.

Histopathological staining remains the fibrosis diagnostic gold standard yet suffers from staining artifacts and variability. Nonlinear optical techniques (e.g., spontaneous fluorescence, Second Harmonic Generation) enhance accuracy but struggle with rapid trace-level detection of fibrosis. To address these limitations, a dual-channel nonlinear optical imaging system with excitation wavelengths at 780 nm and 820 nm was developed, enabling simultaneous spontaneous fluorescence and second-harmonic generation imaging through grid localization. This study applies dual-modality nonlinear imaging to achieve label-free, high-resolution visualization of pulmonary and renal fibrosis at the ECM microstructure scale. Through leveraging this system, it is demonstrated that collagen can be rapidly detected via spontaneous fluorescence at 780 nm, whereas the spatial distribution of collagen fibrils is precisely mapped using Second Harmonic Generation at 820 nm. This approach allows for the rapid and sensitive detection of trace fibrosis in a 5-day unilateral ureteral obstruction mouse model. Additionally, we identify that the elastic fibers, which can also be visualized, provide a foundation for staging diagnosis and delivering accurate and quantitative data for pathological studies and analysis. The research findings underscore the potential of this dual-channel nonlinear optical imaging system as a powerful tool for rapid, precise, and noninvasive fibrosis detection and staging.