- high-resolution terminology - matching measurements at high-resolution
Oxygen flux - instrumental background
Description
Instrumental background oxygen flux, J°O2, in a respirometer is due to oxygen consumption by the POS, and oxygen diffusion into or out of the aqueous medium in the O2k-Chamber. It is a property of the instrumental system, measured in the range of experimental oxygen levels by a standardized instrumental background test. The oxygen regime from air saturation towards zero oxygen is applied generally in experiments with isolated mitochondria and intact or permeabilized cells. To overcome oxygen diffusion limitation in permeabilized fibres and homogenates, an elevated oxygen regime is applied, requiring instrumental background test in the same range of elevated oxygen.
Instrumental background correction eliminates errors by systemic flux compensation, automatically performed by DatLab. If no experimental background test has been performed, the system default values are used, which are a°=-2.0 pmol/(s·ml) for the intercept at zero oxygen concentration, and b°=0.025 for the slope of background flux as a function of oxygen concentration.
Automatic correction for the instrumental background oxygen flux is an essential standard in high resolution respirometry. At the same time an instrumental background experiment is the ultimate test for instrumental performance, evaluating chamber performance after completion of all elements of the Oxygen sensor test. The instrumental background oxygen flux measured at air saturation should reflect the theoretically predicted volume-specific oxygen consumption by the oxygen sensor. The actual agreement using experimental respiration medium provides at the same time a test that excludes microbial contamination of the medium or serves to evaluate any autoxidation processes in newly tested experimental media.
Abbreviation: J°O2
Reference: MiPNet14.06 Instrumental O2 background; Gnaiger_2008_POS; Gnaiger_2001_RespPhysiol
MitoPedia concepts:
MiP concept
MitoPedia methods:
Respirometry
MitoPedia O2k and high-resolution respirometry:
DatLab,
Oroboros QM
- » See MiPNet14.06 Instrumental O2 background for experimental details of the instrumental background test, and for downloading the Excel template for analyzing instrumental background experiments.
- » Compare Chemical background correction of oxygen flux.
MitoPedia O2k and high-resolution respirometry:
O2k-Open Support
O2 slope in the closed and open O2k-Chamber
- In an open chamber of the O2k the liquid phase in the chamber (aqueous medium) is in equilibrium with the atmosphere. All oxygen consumed by the polarographic oxygen sensor (POS) is immediately replaced from the atmosphere. The oxygen signal therefore has to be constant and the (negative) time derivative of the oxygen signal, called "O2 slope uncorr." in DatLab, has to be zero. The background corrected oxygen flux is meaningless for the open chamber situation. This is because the background correction at air saturation subtracts the consumption of oxygen by the sensor from the negative slope, when diffusion into and out of the chamber is zero at air saturation. Therefore, the background-corrected oxygen flux in the open chamber at air saturation is shown as a negative value. To avoid this apparent artefact, the "O2 slope uncorr." is selected to be shown while the chamber is open. Only Graph Layouts that display "O2 slope uncorr." are suitable for assessing the stability of the oxygen signal when the chamber is open. Such Layouts are:
- 01 Calibration show Temp
- 02 Calibration - Background
- In an open chamber of the O2k the liquid phase in the chamber (aqueous medium) is in equilibrium with the atmosphere. All oxygen consumed by the polarographic oxygen sensor (POS) is immediately replaced from the atmosphere. The oxygen signal therefore has to be constant and the (negative) time derivative of the oxygen signal, called "O2 slope uncorr." in DatLab, has to be zero. The background corrected oxygen flux is meaningless for the open chamber situation. This is because the background correction at air saturation subtracts the consumption of oxygen by the sensor from the negative slope, when diffusion into and out of the chamber is zero at air saturation. Therefore, the background-corrected oxygen flux in the open chamber at air saturation is shown as a negative value. To avoid this apparent artefact, the "O2 slope uncorr." is selected to be shown while the chamber is open. Only Graph Layouts that display "O2 slope uncorr." are suitable for assessing the stability of the oxygen signal when the chamber is open. Such Layouts are:
- The observation of a zero flux with an open chamber is an important performance parameter. It indicates that thermal stability and equilibrium of oxygen between the gas and aqueous phases have been reached. Therefore no experiment should be started before a zero "O2 slope uncorr." has been reached with an open chamber. The suggested criterion for signal stability is a "O2 slope uncorr." between -1 pmol/(s mL) and + 1 pmol/(s mL).
