Difference between revisions of "Oxygen calibration - DatLab"

Revision as of 13:34, 11 August 2017

high-resolution terminology - matching measurements at high-resolution

Oxygen calibration - DatLab

Description

O2 calibration

Calibration of the oxygen sensor is a prerequisite for obtaining valid measurements of respiration. Accurate calibration of the oxygen sensor depends on (1) equilibration of the incubation medium with air oxygen partial pressure at the temperature defined by the experimenter; (2) zero oxygen calibration; (3) high stability of the POS signal tested for sufficiently long periods of time; (4) linearity of signal output with oxygen pressure in the range between oxygen saturation and zero oxygen pressure; and (5) accurate oxygen solubility for aqueous solutions for the conversion of partial oxygen pressure into oxygen concentration. The standard oxygen calibration procedure is described below for high-resolution respirometry with the automatic calibration routine by DatLab.

This site is under construction!!

Reference: MiPNet06.03 POS-calibration-SOP

MiPNet19.18D_O2k-Series_G_and_DatLab_6:_Calibration

MiPNet12.20_O2k-calibration_tutorial

MitoPedia methods: Respirometry

MitoPedia O2k and high-resolution respirometry: DatLab

Air calibration

O2k-SOP Air saturation is achieved by stirring the aqueous medium in contact with air in the O2k-chamber without sample, following the procedures below.

1. Add incubation medium into the chambers with an excess volume of at least 0.1 ml above the experimental chamber volume (2 ml) in order to fill the O2k-chamber and injection capillary of the stopper when it is fully inserted (closed). The volume does not have to be accurate, as long as it is above the minimum volume. Switch on the stirrers either during or after addition of the medium.

2. Insert the stoppers slowly to their volume-calibrated position. Suck off excess medium ejected through the injection capillary and collected in the well of the stopper. Then lift the stoppers using the stopper-spacer tool, leaving a gas volume above the liquid phase for final air equilibration.

The central level of the gas phase remains above the rotating stirrer bar, preventing bubbles and foam from being formed which would block gas exchange. To ensure a well defined pO2 in the gas phase, the gas volume has to be renewed (exchanged for air), if the medium was originally not near air saturation. This is achieved simply by fully inserting and re-opening the stopper. Equilibration is a slow process: stability should be reached within one hour (figure below). A stirrer test F9 can be performed during equilibration.

3. After stabilization of the POS signal, the recorded signal at air saturation, R1, is about 1-3 V at Gain 1 and a temperature of 25-37°C. A signal of 1 V corresponds to a signal current of the POS of 1 µA (corresponding to 2 V at Gain 2). Under all experimental conditions, the raw signal must be <10 V.

Continue recording for 3-10 min to check for signal stability. You may proceed at this point with an O2 background test (see below).

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 Accurate calibration of the oxygen sensor depends on (1) equilibration of the incubation medium with air oxygen partial pressure at the temperature defined by the experimenter; (2) zero oxygen calibration; (3) high stability of the POS signal tested for sufficiently long periods of time; (4) linearity of signal output with oxygen pressure in the range between oxygen saturation and zero oxygen pressure; and (5) accurate oxygen solubility for aqueous solutions for the conversion of partial oxygen pressure into oxygen concentration. The standard oxygen calibration procedure is described below for high-resolution respirometry with the automatic calibration routine by DatLab.
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+== Air calibration ==
+O2k-SOP	Air saturation is achieved by stirring the aqueous medium in contact with air in the O2k-chamber without sample, following the procedures below.
+.	Add incubation medium into the chambers with an excess volume of at least 0.1 ml above the experimental chamber volume (2 ml) in order to fill the O2k-chamber and injection capillary of the stopper when it is fully inserted (closed). The volume does not have to be accurate, as long as it is above the minimum volume. Switch on the stirrers either during or after addition of the medium.
+.	Insert the stoppers slowly to their volume-calibrated position. Suck off excess medium ejected through the injection capillary and collected in the well of the stopper. Then lift the stoppers using the stopper-spacer tool, leaving a gas volume above the liquid phase for final air equilibration.
+The central level of the gas phase remains above the rotating stirrer bar, preventing bubbles and foam from being formed which would block gas exchange. To ensure a well defined pO2 in the gas phase, the gas volume has to be renewed (exchanged for air), if the medium was originally not near air saturation. This is achieved simply by fully inserting and re-opening the stopper. Equilibration is a slow process: stability should be reached within one hour (figure below). A stirrer test F9 can be performed during equilibration.
+.	After stabilization of the POS signal, the recorded signal at air saturation, R1, is about 1-3 V at Gain 1 and a temperature of 25-37°C. A signal of 1 V corresponds to a signal current of the POS of 1 µA (corresponding to 2 V at Gain 2). Under all experimental conditions, the raw signal must be <10 V.
+Continue recording for 3-10 min to check for signal stability. You may proceed at this point with an O2 background test (see below).