Intracellular calcium signaling is of paramount importance for epithelial physiology, but capturing and interpreting the heterogeneity of its dynamics at single-cell resolution remains a major research challenge. We report a methodological framework for studying calcium responses in live small intestinal organoids using genetically encoded FRET-based biosensors in combination with unsupervised clustering of calcium traces. Using electroporation, we achieved stable transfection and expression of the FRET-FLIM biosensors (Twitch-2B, Tq-Ca-FLITS) in pig and mouse intestinal organoids. Calcium dynamics were monitored in organoids with both ratiometric and fluorescence lifetime imaging microscopies (FLIM). Pharmacological stimulation showed heterogeneous responses across individual tracked cells. We applied two complementary functional clustering approaches: k-means on B-spline representations of traces, and GPMix, a probabilistic clustering method based on Gaussian processes mixtures. Both enabled grouping of cells by dynamic phenotype without prior assumptions of cell identity. This approach identified distinct temporal profiles, such as rapid responders, delayed activators, or sustained signalers. Crucially, this allowed mapping of each cluster back to its exact spatial position in the organoid image. Such clustering provides an opportunity for dissecting stimulus directionality and wave propagation across the epithelium, as well as for identifying specialized response types that may correspond to particular cell lineages. To validate cluster identities, we aim to multiplex live calcium imaging with immunofluorescence labelling of specific cell type markers, together with guided differentiation of organoids into specific cell types. Presented live cell imaging pipeline demonstrates how advanced clustering of calcium dynamics can transform live-cell imaging into a tool for functional cell type identification aiding studies of the epithelial cell functional heterogeneity in barrier disfunction, inflammatory disease and cell-driven tissue renewal relevant for cancer and regeneration. Supported by European Union, fliMAGIN3D-DN Horizon Europe-MSCA-DN no. 101073507 grant.
Cervellera et al. (Sun,) studied this question.