Browsing by Author "Kao, Joseph P.Y."
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Item Fluorescence Measurement of Pericellular Oxygen(Elsevier Inc., 2016-02-16) Boyman, Liron; Kao, Joseph P.Y.; Leach, Jennie B.; Lederer, W. Jonathan; Williams, George S.B.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.Item Real-time local oxygen measurements for high resolution cellular imaging(Elsevier, 2018-12-05) Boyman, Liron; Williams, George S.B.; Wescott, Andrew P.; Leach, Jennie B.; Kao, Joseph P.Y.; Lederer, W. JonathanSingle-cell metabolic investigations are hampered by the absence of flexible tools to measure local partial pressure of O₂ (pO₂) at high spatial-temporal resolution. To this end, we developed an optical sensor capable of measuring local pericellular pO₂ for subcellular resolution measurements with confocal imaging while simultaneously carrying out electrophysiological and/or chemo-mechanical single cell experiments. Here we present the OxySplot optrode, a ratiometric fluorescent O₂-micro-sensor created by adsorbing O₂-sensitive and O₂-insensitive fluorophores onto micro-particles of silica. To protect the OxySplot optrode from the components and reactants of liquid environment without compromising access to O₂, the micro-particles are coated with an optically clear silicone polymer (PDMS, polydimethylsiloxane). The PDMS coated OxySplot micro-particles are used alone or in a thin (~50 μm) PDMS layer of arbitrary shape referred to as the OxyMat. Additional top coatings on the OxyMat (e.g., fibronectin, laminin, polylysine, special photoactivatable surfaces etc.) facilitate adherence of cells. The OxySplots report the cellular pO₂ and micro-gradients of pO₂ without disrupting the flow of extracellular solutions or interfering with patch-clamp pipettes, mechanical attachments, and micro-superfusion. Since OxySplots and a cell can be imaged and spatially resolved, calibrated changes of pO₂ and intracellular events can be imaged simultaneously. In addition, the response-time (t₀.₅ = 0.7 s, 0–160 mmHg) of OxySplots is ~100 times faster than amperometric Clark-type polarization microelectrodes. Two usage example of OxySplots with cardiomyocytes show (1) OxySplots measuring pericellular pO₂ while tetramethylrhodamine methyl-ester (TMRM) was used to measure mitochondrial membrane potential (ΔΨₘ); and (2) OxySplots measuring pO₂ during ischemia and reperfusion while rhod-2 was used to measure cytosolic [Ca²⁺]ᵢ levels simultaneously. The OxySplot/OxyMat optrode system provides an affordable and highly adaptable optical sensor system for monitoring pO₂ with a diverse array of imaging systems, including high-speed, high-resolution confocal microscopes while physiological features are measured simultaneously.