Multi-parametric O₂ imaging in three-dimensional neural cell models with the phosphorescent probes.

Recent progress in bio-imaging has allowed detailed mechanistic studies of neural cell function in complex 3D tissue models including multicellular aggregates, neurospheres, excised brain slices, ganglia, and organoids. Molecular oxygen (O2 ) is an important metabolite and an environmental parameter which determines the viability and physiological status of neural cells within tissue. Here we describe standard method for monitoring O2 in 3D tissue models using phosphorescence lifetime imaging microscopy (PLIM ) and cell-penetrating O2-sensing probes. The O2 probes can be multiplexed with many conventional fluorescence based live cell biomarkers and also end-point immunofluorescence staining. The multi-parametric O2 imaging method is particularly useful for areas such as stem cell development and differentiation , hypoxia research, neurodegenerative disorders, regeneration of brain tissue, evaluation of new drugs, and development of novel tissue models.