Multicellular spheroids are useful models of mammalian tissue for studies of cell proliferation, differentiation, replacement therapies and drug action. Having a size of 100-500 mu m they mimic in vivo microenvironment and characteristic gradients of O-2, pH and nutrients. We describe the use of cell-penetrating O-2 probes based on phosphorescent Pt-porphyrins to perform high-resolution 2D and 3D mapping of O-2 in spheroid structures by live cell fluorescence imaging technique. Optimised procedures for preparation of neurospheres from cortical neural cells isolated from embryonic rat brain, their staining with the phosphorescent O-2 probes NanO(2) and MM2 and subsequent analysis of oxygenation on different live cell imaging platforms, including widefield and confocal phosphorescence lifetime imaging microscopy (PLIM), conventional confocal and two-photon ratiometric intensity based O-2 detection are presented. This is followed by a series of physiological experiments in which oxygenation patterns of the neurospheres are correlated with culturing conditions (atmospheric hypoxia and hyperoxia, size, growth factors), distribution of stem cells, mature neurons and astrocytes, HIF-2 alpha stabilisation and responses to metabolic stimulation. The O-2 imaging method allows multiplexing with many conventional fluorescent probes to perform multi-parametric imaging analysis of cells in 3D microenvironment It can be applied to other types of spheroids and 3D tissue models. (C) 2013 Elsevier Ltd. All rights reserved.