Imaging of oxygen gradients in giant umbrella cells: An ex vivo PLIM study

O₂ plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O₂ levels are well documented; however, the physiological significance of O₂ microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O₂ probe Pt-Glc and confocal fluorescence microscopy, we visualized O₂ distribution in individual giant (>100-μm) umbrella cells located superficially in the urinary bladder epithelium. We optimized conditions for in vivo phosphorescent staining of the inner surface of the mouse bladder and subsequent ex vivo analysis of excised live tissue. Imaging experiments revealed significant (≤85 μM) and heterogeneous deoxygenation within respiring umbrella cells, with radial O₂ gradients of up to 40 μM across the cell, or ∼0.6 μM/μm. Deeply deoxygenated (5–15 μM O₂) regions were seen to correspond to the areas enriched with polarized mitochondria. Pharmacological activation of mitochondrial respiration decreased oxygenation and O₂ gradients in umbrella cells, while inhibition with antimycin A dissipated the gradients and caused gradual reoxygenation of the tissue to ambient levels. Detailed three-dimensional maps of O₂distribution potentially can be used for the modeling of intracellular O₂-dependent enzymatic reactions and downstream processes, such as hypoxia-inducible factor signaling. Further ex vivo and in vivo studies on intracellular and tissue O₂ gradients using confocal imaging can shed light on the molecular mechanisms regulating O₂-dependent (patho)physiological processes in the bladder and other tissues.