Image Tracing of Inflammatory Intestinal Organoids via Computational Clearing.

Computational clearing (CC) enhances widefield (WF) fluorescence microscopy by suppressing out-of-focus haze and autofluorescence, yielding semi-confocal quality images suitable for segmentation and image-based phenotyping. Here, we propose an "image tracing" workflow for inflammatory mouse intestinal organoids (mIOs) using paired CC and WF images to generate a differential signal (CC - WF). mIOs were derived from intestinal crypts of Lgr5-EGFP stem cell reporter mice and expanded under epidermal growth factor, Noggin, and R-spondin (ENR) conditions. Inflammation was induced by dextran sulfate sodium (DSS) treatment. CC processing enhanced phalloidin-stained apical F-actin and improved EGFP signals by reducing background noise, enabling robust segmentation and quantitative extraction of image morphometrics including area, circularity, and perimeter. CC-WF vectors derived from three-dimensional area-perimeter-circularity plots sensitively captured DSS-induced epithelial disruption analogous to a leaky-epithelium phenotype. Transcriptomic analysis by RNA-seq of DSS-treated mIOs revealed upregulation of inflammatory pathways including TNF-α signaling via NF-κB and IL-6/JAK/STAT3, aligning with microscopy findings. In a proof-of-concept demonstration using phalloidin-stained fluorescence images, ROC analysis of the CC-WF workflow achieved an AUC = 0.95 with 87.5% sensitivity and 92.9% specificity in distinguishing intact from injured mIOs.