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

摘要

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 mu m apart (means +/- S.E.M.). A light response experiment showed realtime 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.