The self-referencing oxygen-selective microelectrode: detection of transmembrane oxygen flux from single cells

S. C. Land, D. M. Porterfield (Lead / Corresponding author), R. H. Sanger, P. J. S. Smith

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Abstract

A self-referencing, polarographic, oxygen-selective microelectrode was developed for measuring oxygen fluxes from single cells. This technique is based on the translational movement of the microelectrode at a known frequency through an oxygen gradient, between known points. The differential current of the electrode was converted into a directional measurement of flux using the Fick equation. Operational characteristics of the technique were determined using artificial gradients. Calculated oxygen flux values matched theoretical values derived from static measurements. A test preparation, an isolated neuron, yielded an oxygen flux of 11.46±1.43 pmol cm-2 s-1 (mean ± S.E.M.), a value in agreement with those available in the literature for single cells. Microinjection of metabolic substrates or a metabolic uncoupler increased oxygen flux, whereas microinjection of KCN decreased oxygen flux. In the filamentous alga Spirogyra greveilina, the probe could easily differentiate a 16.6% difference in oxygen flux with respect to the position of the spiral chloroplast (13.3±0.4 pmol cm-2 s-1 at the chloroplast and 11.4±0.4 pmol cm-2 s-1 between chloroplasts), despite the fact that these positions averaged only 10.6±1.8 μm apart (means ± S.E.M.). A light response experiment showed real-time changes in measured oxygen flux correlated with changes in lighting. Taken together, these results show that the self-referencing oxygen microelectrode technique can be used to detect local oxygen fluxes with a high level of sensitivity and spatial resolution in real time. The oxygen fluxes detected reliably correlated with the metabolic state of the cell.

Original languageEnglish
Pages (from-to)211-218
Number of pages8
JournalJournal of Experimental Biology
Volume202
Issue number2
Publication statusPublished - 1999

Keywords

  • Metabolism
  • Mitochondria
  • Mitochondrial disease
  • Neuron
  • Oxygen flux
  • Oxygen-selective microelectrode
  • Photosynthesis
  • Single cell

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