Fluorescence Measurement of Pericellular Oxygen
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2016-02-16
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Liron Boyman, Joseph P.Y. Kao, Jennie B. Leach, W. Jonathan Lederer, George S.B. Williams, Fluorescence Measurement of Pericellular Oxygen, Biophysical Journal , Volume 110, ISSUE 3, SUPPLEMENT 1, 472a, 2016 , DOI: https://doi.org/10.1016/j.bpj.2015.11.2525
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Abstract
Under normal physiological conditions, the mitochondrial electron transport chain consumes cellular/mitochondrial oxygen (O₂) to generate a proton motive force (ΔΨm) which powers ATP synthesis. Thus, many cellular processes that depend on [ATP] are either directly or indirectly regulated by the availability of O₂ to the cells. Restriction of tissue blood flow disrupts the O₂ supply to cause ischemia, and is detrimental to virtually all cell types. Nevertheless, the cellular, organellar, and molecular processes that enable cells to survive episodes of ischemia remain largely unknown. This is due in part to our inability to control and to accurately measure local pericellular O₂, particularly during high-resolution single cell measurements. Here we present a fluorescent O₂-probe comprising silica-supported O₂ micro sensors integrated in an O₂-permeable silicon matrix. A micro-layer (20-50 μm) of the probe serves as a substrate beneath adherent single cells. This enables us to measure the pericellular partial pressure of O₂ (ppO₂) without disrupting the flow of extracellular solutions. The O₂ probe responds rapidly to abrupt changes of ppO₂ at rates approximately 100 times faster than Clark-type microelectrodes. Since the ppO₂ microsensors and the cell are spatially resolved, calibrated changes of pericellular ppO₂ and intracellular events can be imaged simultaneously with high spatiotemporal resolution. We have used this technique to measure the pericellular ppO₂ of cardiomyocytes, which are extremely vulnerable to O₂ deprivation, owing to their heavy reliance on mitochondrial oxidative phosphorylation to generate ATP. During time-lapse experiments, the ΔΨm in different subcellular regions is monitored. Dynamic cytosolic [Ca²⁺] levels are also measured during electrically stimulated cell contractions and their sensitivity to O₂ is tested. This new probe provides a direct means of measuring pericellular ppO₂ in real-time, and thus opens new avenues to investigate the physiological and pathophysiological responses to hypoxia at the single-cell level.