Molecular oxygen (O2) plays a multitude of important roles in cell and tissue function and (patho)physiology. Real-time quantitative imaging of O2 by phosphorescence quenching method enables detailed mechanistic studies of cell and tissue physiology, responses to hypoxia, drug treatment and other stimuli. We applied high-resolution Phosphorescence Lifetime Imaging Microscopy (PLIM) to study several different 3D tissue models: multi-cellular spheroids, excised animal tissue and cultured organoids. To achieve efficient, stable passive staining of the cells and tissues, we have designed a panel of cell-penetrating phosphorescent nanosensors with variable surface charge and spectral characteristics, and a small molecule O2 probe, Pt-Glc, which provides fast and in-depth staining of most cell models. Using Pt-Glc and PLIM we studied cultures of PC12 (rat pheochromocytoma) and HCT116 (human colon cancer) cell spheroids, intestinal organoids and ex vivo brain, colon, bladder and vessel tissue samples. We showed that cell aggregates of >50 um size are significantly deoxygenated under ambient O2, but remain viable and respond to treatment with compounds affecting metabolism. For the neurospheres from embryonic rat brain, standard protocol was developed for O2 imaging by PLIM, multiplexed with immunofluorescence of cell type, proliferation and death markers. In giant umbrella cells of mouse bladder epithelium we observed marked intracellular gradients of O2 of up to 40-50 mM across the cell or 0.6 mM/mm, which may play physiological roles in tissue function. We found decreased respiration in the colonocytes in dextran sulfate sodium (DSS)-induced colitis mouse colon tissue. These results demonstrate the utility of cell-penetrating O2 probes and PLIM method for life science research.
This work was supported by Science Foundation Ireland (SFI) grants 12/RC/2276 and 13/SIRG/2144.
To cite this abstract:Dmitri Papkovsky, Alexander Zhdanov, Ruslan Dmitriev; High-Resolution Phosphorescence Lifetime Imaging of Oxygen in 3D Tissue Models. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/high-resolution-phosphorescence-lifetime-imaging-of-oxygen-in-3d-tissue-models/. Accessed: November 29, 2022
EMC Abstracts - https://emc-proceedings.com/abstract/high-resolution-phosphorescence-lifetime-imaging-of-oxygen-in-3d-tissue-models/