Talk:Oxygen calibration - DatLab

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Talk:Oxygen calibration - DatLab

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O2k-Open Support

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


O2 calibration is the calibration in DatLab of the oxygen sensor. It is a prerequisite for obtaining accurate 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 calibration routine using instrumental calibration DL-Protocols in DatLab.

Reference: MiPNet06.03 POS-calibration-SOP, MiPNet12.20_O2k-calibration_tutorial

Oxygen calibration - when and how?

Air calibration / Quality control - R1

  • Daily air calibration goes hand in hand with a stirrer test and inspection of the recorded data in DatLab, also called oxygen sensor test. The procedure is decisive for maintaining a high level of O2k quality control as described in QC 1: Oxygen sensor test.
  • Use the air calibration protocol as a guide to generate R1. Main steps:
  • Record the oxygen signal while the chamber is filled with medium and the stopper is in the 'open chamber' position.
  • After equilibration set a mark and call it R1 (named automatically when the DatLab protocol is used)
O2 calibration indicator fields in DatLab 7
O2 calibration indicator fields in DatLab 8
Indicator fields
As a reminder, the calibration indicator fields in the bottom right and bottom left corner of the experiment window are colored yellow after starting an experiment. After opening the calibration window by pressing F5 (or via the top menu bar) and confirming the calibration, the field changes to green (in DatLab 8 as well as in DatLab 7.4).

Zero oxygen calibration - R0

  • Less frequent than air calibration, zero oxygen calibration should be done from time to time over weeks. In addition, zero oxygen calibration is recommended when the experimental workflow requires recording of low oxygen concentrations. It is mandatory after application of a new membrane and O2-sensor service to confirm sensor serviceability.
  • Use the 'air and zero' calibration protocol as a guide to generate R0. Main steps:
  • In the closed chamber remove the oxygen from the medium (chemically by adding sodium dithionite or biologically by adding oxygen consuming sample)
  • Set a mark and call it R0 (named automatically during the DatLab protocol)
Instrumental O2 background test
As described in more detail in MiPNet14.06_Instrumental_O2_background, the instrumental O2 background test is necessary to assess the oxygen fluxes into or out of the respirometer chamber that are not directly related to respiratory activity of the sample. Is is recommended to perform it about once a month. Air calibration and zero oxygen calibration are part of the procedure.

DatLab 8

O2 calibration window manual.png
O2 calibration window calib and active file.png
O2 default calibration window.png

Open the 'O2 calibration window' via selecting 'Calibration' from the top menu bar or by pressing 'F5'.

Together with the oxygen sensor test the air calibration step should be performed on a daily basis prior to starting an experiment. This procedure is vitally important for quality control purposes.

Section 'O2 calibration Chamber A'

  • If an O2 calibration was performed with the same sensor numbers before, the values in this section will be preset according to the earlier calibration. Still, make sure that the values are still valid for the current experiment.
  • Take care to enter the correct O2 solubility factor for your medium. The field 'O2 solubility factor of medium' will be '1.0000' by default as a starting point. Typical media have lower values, e.g. 0.92 for MiR05.

Section 'Air calibration source'

Active file
should be selected when the daily air calibration routine is performed.
It requires the presence of a mark with the name 'R1'. When performing an air calibration guided by the DatLab protocol 'Instrumental procotol: air calibration' the mark name will be set to 'R1' automatically. If not, create a mark and change its name to 'R1' by clicking on its top blue bar in the graph.
After changing the mark R1 during a calibration, press 'Update' load the new value.
Calibration file
is preselected whenever DatLab finds calbration values that were applied earlier. Check and confirm the values.
Alternatively, another file recorded with the same sensor number and containing a mark 'R1' can be loaded.
is pre-selected when DatLab 8 does not find values from a previous O2 calibration. Default values are shown and can be edited manually.

Section 'Zero calibration source'

  • The same options are available: 'Manual' as a default if no other values are available and for manual input; 'Calibration file' shows where preset values come from. Alternatively, values from another file can be loaded. The option 'Active file' requires a mark named R0 in the active measurement. The DatLab-protocol: 'air and zero' provides guidance.
  • In contrast to the 'air calibration' step, it is enough to perform zero calibration from time to time. Therefore 'Calibration file' will be applied more often.

Section 'O2 calibration values'

  • Values computed from the input sources (FM, R1 and R0) above are displayed but cannot be edited in this section.
  • Click 'Show details' for a more detailed compilation of values used to compute an oxygen concentration from the raw signal received from the POS sensor.
  • 'Copy to clipboard' for data analysis outside of DatLab8. We recommend to use Excel templates provided together with the O2k instrument for long-time record of R1 and R0 to observe sudden changes as hints for errors.


Reset to defaults O2 solubility factor FM is set to '1.0000', R1 to '2.0000', Temperature to '37.00', Barometric pressure to '100.00' and R1 to '0.0000'
Values are applied to the active measurement or the open file without closing the window.
During active measurement a dialog box 'Default calibration' pops up asking if the current O2 calibration parameters should be used as default for the respective chamber. Selecting 'Yes' prompts DatLab 8 to write updated values (FM, R1 or R0) to the default settings, from where they will be loaded as default settings for the next experiment.
OK same as 'Apply', the window is closed.

Default settings

  • If a POS sensor was not calibrated before, the calibration is set to 'Manual' with values that are also accessible via 'Reset to defaults'-button. These values are not intended for use in experiments, they will show an arbitrary O2 concentration, but it is enough to see the blue line in the graph and perform O2 calibration.
  • During an active measurement and after selecting "Apply" or "OK", the user will be asked if the O2 calibration parameters should be used as default. Selecting 'Yes' will prompt DatLab to use the parameters for future experiments. The name of the calibration file will be displayed next to 'Calibration file' checkbox. DatLab 8 makes sure that the sensor number of the saved values corresponds to the sensor number in use. It does not care about other settings. It is the user's responsibility to make sure that
  • solubility factor FM
  • medium
  • and experimental temperature
of the calibration experiment correspond with the experiment the default values are used for.

DatLab 7.4

DLP O2-calibration air and zero.png
Calibration window steps.png

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