Sc. Land et al., The self-referencing oxygen-selective microelectrode: Detection of transmembrane oxygen flux from single cells, J EXP BIOL, 202(2), 1999, pp. 211-218
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.