Gradients of Po2 between capillary blood and mitochondria are the driving force for diffusional O2 delivery in tissues. Hypoxic microenvironments in tissues that result from diffusional O2 gradients are especially relevant in solid tumors because they have been related to a poor prognosis. To address the impact of tissue O2 gradients, we developed a novel technique that permits imaging of intracellular O2 levels in cultured cells at a subcellular spatial resolution. This was done, with the sensitivity to O2 ≤3%, by the O2-dependent red-shift of green fluorescent protein (AcGFP1) fluorescence. Measurements were carried out in a confluent monolayer of Hep3B cells expressing AcGFP1 in the cytoplasm. To establish a 2-dimensional O2 diffusion model, a thin quartz glass slip was placed onto the monolayer cells to prevent O2 diffusion from the top surface of the cell layer. The magnitude of the red-shift progressively increased as the distance from the gas-cover slip interface increased. It reached an anoxic level in cells located at ~220 µm and ~690 µm from the gas-cover slip boundary at 1% and 3% gas phase O2, respectively. Thus, the average O2 gradient was 0.03 mmHg/µm in the present tissue model. Abolition of mitochondrial respiration significantly dampened the gradients. Furthermore, intracellular gradients of the red-shift in mitochondria targeted AcGFP1 in single Hep3B cells suggest that the origin of tissue O2 gradients is intracellular. Findings in the present 2-dimensional O2 diffusion model support the crucial role of tissue O2 diffusion in defining the O2 microenvironment in individual cells.
- oxygen sensing
- Copyright © 2010, American Journal of Physiology - Cell Physiology