Imaging cell and tissue O₂ by TCSPC-PLIM

We describe a technique of imaging tissue oxygen using phosphorescence based probes and TCSPC-PLIM method. Included is a brief overview of the significance of biological oxygen imaging, the theory behind the phosphorescence quenching method, the main O2 sensitive probes (mostly intracellular, cellpermeable) and imaging modalities currently available, highlighting their merits and limitations. In the practical part, the live cell microscopy imaging and TCSPC-PLIM hardware and software are described, along with the detailed experimental procedures of preparation of tissue samples, their staining with intracellular O2 probes, acquisition of PLIM images and their processing to produce 2D and 3D maps of O2 concentration. Several examples demonstrate practical use of O2 imaging with different models of mammalian tissue, including cell mono-layers (2D model), multicellular spheroids, scaffold structures and tissue slices (3D models). Physiological experiments and multi-parametric analysis of these samples with some other fluorescent imaging probes are also presented.