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

A self-referencing, polarographic, oxygen-selective microelectrode was deve loped for measuring oxygen fluxes from single cells. This technique is base d 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 deter mined using artificial gradients. Calculated oxygen flux values matched the oretical values derived from static measurements. A test preparation, an is olated neuron, yielded an oxygen flux of 11.46+/-1.43 pmol cm(-2) s(-1) (me an +/- S.E.M.), a value in agreement with those available in the literature for single cells. Microinjection of metabolic substrates or a metabolic un coupler increased oxygen flux, whereas microinjection of KCN decreased oxyg en flux. In the filamentous alga Spirogyra greveilina, the probe could easi ly differentiate a 16.6 % difference in oxygen flux with respect to the pos ition of the spiral chloroplast (13.3+/-0.4 pmol cm(-2) s(-1) at the chloro plast and 11.4+/-0.4 pmol cm(-2) s(-1) between chloroplasts), despite the f act 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 oxyge n flux correlated with changes in lighting. Taken together, these results s how that the self-referencing oxygen microelectrode technique can be used t o detect local oxygen fluxes with a high level of sensitivity and spatial r esolution in real time. The oxygen fluxes detected reliably correlated with the metabolic state of the cell.