Zero calibration

From Bioblast

high-resolution terminology - matching measurements at high-resolution

Zero calibration


Zero calibration is, together with air calibration, one of the two steps of the POS calibration. It is performed in the closed chamber after all the oxygen has been depleted by the addition of dithionite or by respiration of imt or cells. Any incubation medium can be used for zero calibration with dithionite or sample. Unlike air calibration, it is not necessary to perform a zero calibration on each experimental day. After performing a zero calibration, it is recommended not running other experiments on the same day. Even after standard cleaning of the O2k-chambers, there might be residual amounts of reduced dithionite in the chamber, affecting the oxygen flux in subsequent experiments performed on the same day.

Abbreviation: R0

Reference: MiPNet06.03 POS-calibration-SOP, O2 calibration - DatLab

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MitoPedia O2k and high-resolution respirometry: O2k-Open Support 


A zero calibration should be performed
A zero calibration has to be performed
  • When measurements are performed at low oxygen (<10 Β΅M).
  • Bracketing zero calibrations are required when measurements are performed near the limit of detection at oxygen concentrations close to 5 nM (0.005 Β΅M).
  • When a new or serviced POS is used for the first time.
  • When a new membrane was applied to the sensor.
  • When the gain is changed.
  • When a new OroboPOS-Connector is used.

Incubation medium for zero O2 calibration with dithionite

Any incubation medium can be used for zero calibration with dithionite. MiR05 or a highly concentrated phosphate buffer are needed when using dithionite for measuring of instrumental background oxygen flux, to avoid secondary oxygen-consuming reactions after a step titration of dithionite. For zero calibration, however, an excess dithionite concentration is applied which consumes all dissolved oxygen in the medium, and secondary reactions are not an issue.

Zero current

Each polarographic oxygen sensor has a zero current, i.e., a current [Β΅A] that flows even at an oxygen concentration of zero. This zero oxygen signal is taken into account by the zero calibration and can be expressed as % of the raw signal at air saturation. New POS have a zero signal below 2 % of the signal at air saturation. For accurate calibration, the zero signal must be stable, which may be the case for zero currents up to 5 % of the signal at air saturation. The stability of the zero current can be assessed by keeping track of calibration values using the "O2k-Calibration-list.xls" template.
A high zero current might not only be caused by the POS, but can also be caused by the O2k-Main Unit, the OroboPOS-Connector, or by dirty electrical connections between those parts. If the zero current is too high a leak current test helps to locate the problem.
Β» Further details: High signal at zero oxygen


Β» MiPNet06.03 POS-calibration-SOP

Keywords: Oxygen signal


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MitoPedia methods: Respirometry 

MitoPedia O2k and high-resolution respirometry: DatLab 

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