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 



Description

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 the raw signal from the POS should be stable and around 2 (acceptable between 1 and 3).
  • 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).
  • The raw signal from the POS should be stable and < 2% of the signal at air saturation (up to 5% is acceptable).
  • 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

DatLab 8: Data required for O2 calibration: R1 for air calibration and R0 for zero calibration
DatLab 8: Calibration from file or from active file

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'

  • Sensor number and medium are displayed according to user input before starting a measurement. They can be updated here.
  • 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. If an O2 calibration was performed (and saved as default) before or loaded from a file, the value will be preset according to the earlier calibration. DatLab 8 accepts earlier calibrations only if they were performed with the same sensor. Still, make sure that the values are still valid for the current experiment.

Section 'Air calibration source'

Active file
should be selected when the daily air calibration routine is performed.
A mark with the name 'R1' is required. When performing an air calibration guided by the DatLab protocol 'Instrumental protocol: 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' to load the new value.
Calibration file
is preselected whenever DatLab finds calibration 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.
Manual
is preselected when DatLab 8 does not find values from a previous O2 calibration. Default values are shown and can be edited manually.
Default values of option 'Manual' provide a starting point but are not appropriate for use in experiments.

Section 'Zero calibration source'

  • The same options are available: 'Manual', 'Calibration file' and 'Active file'. The latter 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.
  • Keep an eye on the value 'O2 slope neg.' (orange arrow). It should be within the range of -1 to 1 pmol∙s-1∙mL-1 as a quality criterion.
  • Click 'Show details' for a more detailed compilation of values used to compute the 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.

Buttons

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'. These values give a starting point but should not be use for experiments.
Apply Values are applied to the active measurement or the open file without closing the window.
OK same as 'Apply', the window is closed.
O2 default calibration window.png

Default settings

  • After selecting "Apply" or "OK" and during an active measurement, the user will be asked if the O2 calibration parameters should be used as default. Selecting 'Yes' will prompt DatLab to use the parameters (FM, R1, R0) 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 is the user's responsibility to make sure that parameters like
  • solubility factor FM
  • medium
  • and experimental temperature
of the calibration experiment correspond with the experiment the default values shall be used for.



DatLab 7.4

DatLab 7: Data required for O2 calibration: R1 for air calibration and R0 for zero calibration
DatLab 7: O2 calibration window

The O2 calibration window is accessible via selecting 'Calibration > A: Oxygen, O2' from the top menu bar, via pressing 'F5' or by double-clicking the blue box 'Y1' right to the plot.

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.

Calibration source 'Default'

If a O2 calibration was confirmed by pressing 'Calibrate and copy to clipboard' in the past, DatLab 7 provides theses values as a default, indicating date and time when the calibration was performed. DatLab 7 does not check if the sensor or instrument numbers match, it does not care about experimental conditions. It is the user's responsibility to make sure that experimental conditions (like temperature or solubility factor of the medium) and of preloaded settings fit the experiment they are used for.

Calibration source 'Copy from file'

A *.DLD file can be selected to load calibration values. The file may contain any type of O2k experiment. It is important that a proper O2 calibration has been conducted (including confirmation of 'Calibrate and copy to clipboard') before saving the file.

Calibration source 'Active file'

As soon as you start to select a mark for air or zero calibration, the field 'Calibration source' changes to 'Active file'. It is recommended to generate calibration data guided by the DatLab Protocol ('Protocols > A: Run Protocol / Set O2 limit Y > Instrumental > O2 calibration > O2 calibration air' or 'O2 calibration air and zero'). Following the SOPs explained therein, the marks will be named automatically. Select the marks from the drop-down menu for R1 and R0 and confirm with 'Calibrate and copy to clipboard'.

  • Keep an eye on the value 'O2 slope neg.' (orange arrow). It should be within the range of -1 to 1 pmol∙s-1∙mL-1 as a quality criterion.
  • If only R1 was recorded during the daily calibration routine, R0 will be taken from the latest past calibration (see section Calibration source 'default').

Other buttons and fields

POS #
A field to edit the sensor number (not recommended).
Details
This tab next to 'Signal' in the third line shows a more detailed compilation of values used to compute the oxygen concentration from the raw signal received from the POS sensor.
Reset to system default
This option provides values as a starting point. They 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.



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