Live Fluorescence Lifetime Imaging Microscopy (FLIM) of the Epithelial Polarity and Cell-Specific Segmentation in the Small Intestinal Organoids.

Apical-basal polarity is essential for gastrointestinal epithelium function, where the apical membrane mediates nutrient absorption, host-microbiota interactions, and pathogen defense. However, conventional intestinal organoid culture displays an "apical-in" topology, limiting functional research with respect to the apical membrane. F-actin labeling with fluorescent phalloidin, a well-established apical marker, is also incompatible with live imaging. To address these limitations, in this chapter we describe an extracellular matrix (ECM) removal protocol to achieve "apical-out" topology in porcine intestinal organoid model and further validated WGA and Nile Red live imaging tracers enabling discrimination between apical-out (AO) and basal-out (BO) organoids. Subsequently, using integrated computational pipelines (automatic nuclei segmentation and single-nucleus phasor-based lifetime analysis), we can quantify organoid proliferative states at single-nucleus resolution following BrdU and Hoechst 33342 staining. Using the analysis of Hoechst 33342 fluorescence lifetime, we demonstrate that both organoid topologies exhibit intrinsic proliferative heterogeneity, but overall proliferative capacity is topology-dependent: apical-out organoids displayed decreased proliferation, whereas basal-out organoids remain highly proliferative.