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 C _6h 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 the organization of C _6h symmetry. When both hemalum and eosin are used for H&E staining, the dye molecules interact and align noncentrosymmetrically with C ₆ 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 $R={\chi }_{zzz}^{\left(2\right)}$ / ${\chi }_{zxx}^{\left(2\right)}$ 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.