ABSTRACT The visualization of collagen fiber dynamics in skin tumor infiltration is crucial in oncological research, but conventional imaging faces challenges: Second harmonic generation (SHG) microscopy struggles with fine collagen resolution, while super‐resolution microscopy (SRM) relies on fluorescent labeling. To address these limitations, we present a label‐free multimodal super‐resolution imaging method that combines SHG with point‐scanning structured illumination microscopy (SHG‐SIM), allowing high‐resolution imaging of collagen fibers in skin tissue. Two‐photon excitation fluorescence microscopy (2PEF) was used to localize collagen fibers in the tumor‐infiltrated area. This integrated approach enables precise identification of changes in collagen fiber organization within tumors. To improve quantitative analysis, a novel pixel‐level area (PLA) recognition algorithm is applied to efficiently quantify collagen fiber areas inside and outside the tumor, offering critical insights into tumor progression. A large field‐of‐view stitching algorithm ensures accurate image registration and generates super‐resolution stitched images with an extended field of view. This comprehensive system is a powerful tool for evaluating collagen fiber expression in skin tumor infiltration, with potential applications in tumor microenvironment research, clinical diagnosis, and treatment.
Wang et al. (Tue,) studied this question.