Mapping peritumoral collagen fiber directionality in solid tumors may assist in determining cancer progression and support more personalized prognoses. However, existing microscopy techniques are often limited by a restricted field of view, high cost, or incompatibility with paraffin-treated tissues. Computational scattered light imaging (ComSLI) is a cost-effective whole-slide microscopy technique that reveals fiber orientations independent of sample preparation. Using glioma, colorectal, and head and neck cancer samples, we show for the first time that ComSLI maps fiber orientations in paraffin-treated tumor tissues, visualizes tumor growth pathways and desmoplastic reactions, and allows the study of collagen orientations relative to tumor boundaries.

Collagen forms dense fibre networks in the human body, with the organisation directly influencing tissue mechanics and function in health and disease. A good understanding of this relation requires proper imaging techniques for visualising the dense collagen network. Previously, computational scattered light imaging was employed as a fast and easy-to-implement technique to retrieve the orientations of multi-directional fibres in various tissue samples, but the fibre orientations were not yet validated quantitatively in regions containing collagen fibres. In this study, we validate the in-plane orientations of fibres in collagen-containing tissues (rat tendon and bone sections) determined with computational scattered light imaging by performing comparative measurements with polarimetric second harmonic generation microscopy. For rat tendon, sections with and without hematoxylin-and-eosin staining, folded tendon layers, and obliquely cut sections were investigated. Similar fibre orientations were obtained with both techniques in both tissues, with the highest degree of similarity found for in-plane, unidirectional fibres in the tendon sections. The techniques were able to retrieve the orientations of multi-directional crossing fibres in folded rat tendon layers, and results were found to be unaffected by staining. While polarimetric second harmonic generation microscopy provides high resolution and ultrastructural information on collagen, computational scattered light imaging provides large field of view measurements with micrometre resolution.

Mapping the brain’s fiber network is crucial for understanding its function and malfunction, but resolving nerve trajectories over large fields of view is challenging. Here, we show that computational scattered light imaging (ComSLI) can map fiber networks in histology independent of sample preparation, also in formalin-fixed paraffin-embedded (FFPE) tissues including whole human brain sections. We showcase this method in new and archived, animal and human brain sections, for different sample preparations (in paraffin, deparaffinized, various stains, unstained fresh-frozen). We convert microscopic orientations to microstructure-informed fiber orientation distributions (μFODs). Adapting tractography tools from diffusion magnetic resonance imaging (dMRI), we trace axonal trajectories revealing white and gray matter connectivity. These allow us to identify altered microstructure or deficient tracts in demyelinating or neurodegenerating pathology, and to show key advantages over dMRI, polarization microscopy, and structure tensor analysis. Finally, we map fibers in non-brain tissues, including muscle, bone, and blood vessels, unveiling the tissue’s function. Our cost-effective, versatile approach enables micron-resolution studies of intricate fiber networks across tissues, species, diseases, and sample preparations, offering new dimensions to neuroscientific and biomedical research.