Multi-Parametric Imaging of Hypoxia and Cell Cycle in Intestinal Organoid Culture

Dynamics of oxygenation of tissue and stem cell niches are important for understanding physiological function of the intestine in normal and diseased states. Only a few techniques allow live visualization of tissue hypoxia at cellular level and in three dimensions. We describe an optimized protocol, which uses cell-penetrating O₂-sensitive probe, Pt-Glc and phosphorescence lifetime imaging microscopy (PLIM), to analyze O₂ distribution in mouse intestinal organoids. Unlike the other indirect and end-point hypoxia stains, or point measurements with microelectrodes, this method provides high-resolution real-time visualization of O₂ in organoids. Multiplexing with conventional fluorescent live cell imaging probes such as the Hoechst 33342-based FLIM assay of cell proliferation, and immunofluorescence staining of endogenous proteins, allows analysis of key physiologic parameters under O₂ control in organoids. The protocol is useful for gastroenterology and physiology of intestinal tissue, hypoxia research, regenerative medicine, studying host-microbiota interactions and bioenergetics.