Light-sheet fluorescence microscopy of optically cleared human head-and-neck organs: three-dimensional tissue assessment and histological correlation

Conventional histology provides detailed cellular information but only a partial view of the three-dimensional (3D) organisation of head-and-neck tissues. We investigated whether optical tissue clearing combined with light-sheet fluorescence microscopy (LSFM) could visualise the microanatomy of large head-and-neck specimens and relate volumetric features to conventional histology. Non-neoplastic human palatine tonsil, cervical lymph node, tongue and parotid gland specimens were cleared with a solvent-based protocol, labelled with small-molecule dyes and imaged by LSFM, with haematoxylin and eosin (H&E) histology as a morphological reference. LSFM visualised the characteristic architecture of each tissue, including lymphoid follicles and crypt epithelium in tonsil, cortex, paracortex and follicles in lymph node, epithelium and intrinsic muscle fibres in tongue, and lobules, ducts and adipocytes in parotid. Following structures through successive optical sections revealed their continuity and branching over long distances, information not available from single sections. Tongue epithelial measurements were comparable with H&E, and segmentation enabled volumetric quantification of structures such as the tongue epithelium and parotid adipocytes. Optical clearing and LSFM thus provide a feasible single workflow for volumetric investigation of human head-and-neck microanatomy. These data establish a 3D morphological reference with potential applications in cancer research and staging, computational image analysis and anatomical education, to be further developed through larger donor cohorts and specific labelling.