Background & AimsTissue-clearing and three-dimensional (3D) imaging techniques aid clinical histopathological evaluation; however, further methodological developments are required before use in clinical practice.MethodsWe sought to develop a novel fluorescence staining method based on the classical periodic acid-Schiff stain. We further attempted to develop a 3D imaging system based on this staining method and evaluated whether the system can be used for quantitative 3D pathological evaluation and deep learning–based automatic diagnosis of inflammatory bowel diseases.ResultsWe successfully developed a novel periodic acid–FAM hydrazide (PAFhy) staining method for 3D imaging when combined with a tissue-clearing technique (PAFhy-3D). This strategy enabled clear and detailed imaging of the 3D architectures of crypts in human colorectal mucosa. PAFhy-3D imaging also revealed abnormal architectural changes in crypts in ulcerative colitis tissues and identified the distributions of neutrophils in cryptitis and crypt abscesses. PAFhy-3D revealed novel pathological findings including spiral staircase-like crypts specific to inflammatory bowel diseases. Quantitative analysis of crypts based on 3D morphologic changes enabled differential diagnosis of ulcerative colitis, Crohn’s disease, and non-inflammatory bowel disease; such discrimination could not be achieved by pathologists. Furthermore, a deep learning–based system using PAFhy-3D images was used to distinguish these diseases The accuracies were excellent (macro-average area under the curve = 0.94; F1 scores = 0.875 for ulcerative colitis, 0.717 for Crohn’s disease, and 0.819 for non-inflammatory bowel disease).ConclusionsPAFhy staining and PAFhy-3D imaging are promising approaches for next-generation experimental and clinical histopathology.
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