Mazeika, V. | bioRxiv Science

bioRxiv2025

Collagen fibre organisation revealed with computational scattered light imaging and polarimetric second harmonic generation microscopy

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 orientation of multi-directional collagen fibres, but results were not yet validated quantitatively. In this study, we validate the in-plane orientations of collagen fibres determined with computational scattered light imaging by performing comparative measurements with polarimetric second harmonic generation microscopy on rat tendon and bone sections. 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 collagen 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.

bioengineering↗

bioRxiv2025

Polar organization of H&E dyes in histology tissue revealed by polarimetric nonlinear microscopy

Structural organization of harmonophores used in hematoxylin (H) and eosin (E) staining is studied with polarimetric multimodal second-harmonic generation (SHG), third-harmonic generation (THG) and multiphoton excitation fluorescence (MPF) microscopy in rat tail tendon histology sections. The polarimetric microscopy imaging reveals that hemalums (complexes of hematoxylin and aluminum) are well aligned with C6h symmetry along the collagen fibers in H-stained tissue, while eosin Y is partially aligned along the fibers in E-stained tissue and also follows organization of C6h symmetry. When both hemalum and eosin are used for H&E staining, the dye molecules interact and align noncentrosymmetrically with C6 symmetry along the collagen fibers, while the stained nuclei appear isotropically organized. The polar alignment of the hemalum and eosin complexes increases the achiral second order susceptibility tensor component ratio [Formula] in H&E-stained tissue. The alignment of hemalum and eosin molecules, and their complexes in collagenous tissue, must be considered in nonlinear microscopy and polarimetric analysis of H&E-stained histopathology.

biophysics↗