Okkelman Irina A, Foley Tara, Papkovsky Dmitri B, Dmitriev Ruslan I
Laboratory of Biophysics and Bioanalysis, School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland.
Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland.
Adv Exp Med Biol. 2017;1035:85-103. doi: 10.1007/978-3-319-67358-5_6.
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 O-sensitive probe, Pt-Glc and phosphorescence lifetime imaging microscopy (PLIM), to analyze O 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 O 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 O control in organoids. The protocol is useful for gastroenterology and physiology of intestinal tissue, hypoxia research, regenerative medicine, studying host-microbiota interactions and bioenergetics.
组织和干细胞微环境的氧合动力学对于理解正常和疾病状态下肠道的生理功能至关重要。只有少数技术能够在细胞水平和三维空间实时可视化组织缺氧情况。我们描述了一种优化方案，该方案使用细胞穿透性氧敏感探针Pt-Glc和磷光寿命成像显微镜（PLIM）来分析小鼠肠道类器官中的氧分布。与其他间接和终点缺氧染色或微电极点测量不同，该方法可提供类器官中氧的高分辨率实时可视化。与传统荧光活细胞成像探针（如基于Hoechst 33342的细胞增殖FLIM分析）以及内源性蛋白质的免疫荧光染色进行多重检测，能够分析类器官中氧控制下的关键生理参数。该方案对于肠道组织的胃肠病学和生理学、缺氧研究、再生医学、研究宿主-微生物群相互作用和生物能量学都很有用。
