Novel histological observation method using the scattered light of unstained collagen sections

Collagen fibers are significant constituents of connective tissues such as the skin, bone, and tendons. Their morphology and alignment are essential for understanding tissue function and pathology. Collagen fibers are conventionally observed using optical and electron microscopy, and Second-harmonic generation microscopy. This study introduces a novel microscopic imaging technique, Scattering Angle-Resolved Bioimaging (SARB), which utilizes the scattering properties of unstained specimens. The SARB is based on a conventional optical microscope with a structured illumination module that projects a checker pattern onto the sample. This allows the acquisition of images separated by the scattering angle, enabling the visualization of the scattering characteristics. We applied SARB and electron microscopy to thin, unstained formalin-fixed, paraffin-embedded (FFPE) sections of mouse skin. SARB successfully identified the location of collagen fiber bundles and revealed the scattering information within them. Importantly, these scattering patterns correlated with the orientation of the collagen fibrils, as observed in Transmission Electron Microscopy images. The use of unstained FFPE sections simplifies sample preparation, offering a practical advantage. Additionally, SARB provides access to fine structural information that is often difficult to obtain using conventional optical microscopy. These findings suggest that SARB has strong potential as a label-free, morphology-sensitive imaging method for evaluating fibrosis.