The self-referencing oxygen-selective microelectrode: detection of transmembrane oxygen flux from single cells.
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 microm 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.
Land
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BioCurrents Research Center, Marine Biological Laboratory, Woods Hole, MA 02543, USA.
Porterfield
D M
DM
Sanger
R H
RH
Smith
P J
PJ
D000818:Animals; D001048:Aplysia / Q000378:metabolism; D000460:Chlorophyta / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism; D066298:In Vitro Techniques; D008839:Microelectrodes; D009474:Neurons / Q000378:metabolism; D010100:Oxygen / Q000032:analysis* / Q000378:metabolism
D010100:Oxygen