Oxygen calibration - DatLab

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

Description

DatLab 8: O₂ calibration is the calibration in DatLab of the oxygen sensor (air calibration and zero calibration). It is a prerequisite for obtaining accurate measurements of respiration.

DatLab 7: O₂ calibration

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

Last update: 2024-10-24

DatLab converts the oxygen signal from the Polarographic oxygen sensor (POS) to oxygen concentration (µM) during calibration, this comprises an Air calibration (R1) and a Zero calibration (R0).

Accurate calibration of the oxygen sensor depends on (1) equilibration of the incubation medium with the oxygen partial pressure of air 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.

Instrumental DatLab Protocols guide the O2k operator step-by-step through the calibration procedure, adding automatically the event names and mark names set during the calibration run.

Air calibration: O₂ sensor test and calibration at air saturation: performed routinely every day before starting experimental measurements.

DL8: O2_calibration_air.dld8 (DatLab8\DL-Protocols\Instrumental\O2 calibration)

DL7: O2-calibration_air.DLP (DatLab\DL-Protocols\Instrumental\O2 calibration)

Air and zero calibration: O₂ sensor test and calibration at air saturation and zero oxygen.

DL8: O2_calibration_air_and_zero.dld8 (DatLab8\DL-Protocols\Instrumental\O2 calibration)

DL7: O2-calibration_air and zero.DLP (DatLab\DL-Protocols\Instrumental\O2 calibration)

O₂ calibration in DatLab 8

Air calibration

1. Mark: Mark a section of the experiment at air saturation, when signal stability is reached. If a DL8-Protocol is used, this mark will be recognized as R1 and the ‘O₂ calibration’ window will open automatically. Otherwise it can be opened through the software menu 'Calibration' (F5).

2. Apply: To apply the air calibration in the ‘O₂ calibration’ window:

If not done previously, enter the name of experimental incubation medium and the corresponding O₂ solubility factor of the medium. For more info on this factor: MiPNet06.03 POS-calibration-SOP.

Select Active file as ‘Source of air calibration’ (if a protocol mark R1 is recognized, this will be automatic). The average current recorded over the marked section (POS signal: recorded [µA]) and the corresponding signal stability displayed as the uncorrected negative slope of the signal (O₂ slope neg [pmol∙s⁻¹∙mL⁻¹]) will be displayed below. Temperature and barometric pressure are displayed as measured over the marked section.

Left click on Apply. After clicking on this button all changes in the entire calibration window are applied. The entire plot of oxygen concentration is re-calibrated [µM = nmol/mL], and the corresponding negative slope or volume-specific oxygen flux [pmol∙s⁻¹∙mL⁻¹] is now based on this new calibration. Click on Copy to clipboard before closing the calibration window to copy the calibration parameters for entry into the spreadsheet "Template O2 calibration.xlsx".

Zero calibration

1. Mark: Mark a section of the experiment after full depletion of oxygen, when signal stability is reached. If a DL8-Protocol is used, this mark will be recognized as R0 and the ‘O₂ calibration’ window will open automatically. Otherwise it can be opened through the software menu 'Calibration' (F5).

2. Apply: Apply the zero calibration in the ‘O₂ calibration’ window:

If not done previously, enter the name of experimental incubation medium and the corresponding O₂ solubility factor of the medium. For more info on this factor: MiPNet06.03 POS-calibration-SOP.

Select Active file as ‘Source of zero calibration’ (if a protocol mark R0 is recognized, this will be automatic). The average current recorded over the marked section (POS signal: recorded [µA]) and the corresponding signal stability displayed as the uncorrected negative slope of the signal (O₂ slope neg [pmol∙s⁻¹∙mL⁻¹]) will be displayed below. The stable zero signal, R0, should be <2 % of the signal at air saturation R1, but <5 % is acceptable. Most importantly, the zero signal must be stable. Temperature and barometric pressure are displayed as measured over the marked section and are without influence on the calibration calculations for zero oxygen.

Left click on 'Apply'. After clicking on this button all changes in the entire calibration window are applied. The entire plot of oxygen concentration is re-calibrated [µM = nmol/mL], and the corresponding negative slope or volume-specific oxygen flux [pmol∙s⁻¹∙mL⁻¹] is now based on this new calibration. Click on Copy to clipboard before closing the calibration window to copy the calibration parameters for entry into the spreadsheet "Template O2 calibration.xlsx".

Oxygen calibration in instrumental and experimental DatLab files

It is common practice to save instrumental calibration tests in separate DatLab files, which include (1) air calibration and zero oxygen calibration in the same calibration file, or (2) air calibration and zero oxygen calibration in different calibration files.

1. Instrumental run and DatLab connected: If calibration marks (R1, R0) are set and calibration parameters are applied in the ‘O₂ calibration’ window while DatLab is connected to the O2k during an instrumental calibration run (Active file), a pop-up message will ask the user if this should be the default calibration for the sensor. If yes is selected, the calibration parameters are automatically transferred to the next experimental DatLab files. Therefore, when a new DatLab file is recorded at a later time, the most recent calibration parameters are automatically applied in the new DatLab file — independent of maintaining the O2k in operation or switching it off, and independent of maintaining DatLab active or re-opening it after exit.

2. Importing of calibration parameters from file: Calibration parameters for air calibration, zero oxygen calibration, or both can also be imported from a selected DatLab file. This is particularly important, if a zero calibration is performed after completion and saving an experimental file. This can be done by selecting Calibration file as source of air or zero calibration, and choosing the desired instrumental calibration file by clicking on the folder icon. An error message will be displayed if the sensor number of the select file does not match the one of the current file. Left click on Apply.

3. Inserting calibration parameters manually: A single calibration value (R1 or R0) can be also edited manually by selecting Manual as source of air or zero calibration and entering the desired value. Left click on Apply.

Real-time vs. disconnected

DatLab uses calibration values applied in real-time (connected to the O2k, recording data) as default values for future experiments. When calibration values are edited in the disconnected mode, they apply only to the current file and will not be used as a new default in experiments. This allows to re-calibrate old files without overwriting the current default values for calibration. Ideally, calibration values that should be used as new defaults are applied in real-time when the experiment is still running. If the DatLab-calibration is performed after disconnecting, these calibration parameters can be read into other DatLab files using Calibration file as source of air or zero calibration and apply.

Before disconnecting the O2k from DatLab, calibration information is automatically saved and available upon re-connecting the O2k, even if you exit DatLab and start the program again. The current calibration parameters are displayed when opening the O₂ calibration window [F5].

O₂ calibration in DatLab 7

Air calibration

1. Select Graph layout:

Go to menu Layout, check O2 and from Standard layouts select 01 Calibration show Temp.

This is typically the first layout used after switching on the O2k. Oxygen concentration (blue plots, left Y-axis) and O₂ slope (not corrected for instrumental background; red plots, right Y-axis) are displayed on the top graph (left O2k-chamber), and middle graph (right O2k-chamber). The third graph (bottom) shows the block temperature on the left Y-axis and the Peltier power on the right Y-axis. Only when both temperature and Peltier power are constant, the chambers have reached thermal equilibrium. The next step is to observe equilibration of the oxygen signal with a defined gas phase above the stirred aqueous phase ('open' chamber; usually with air as the first step) to perform an oxygen calibration.

2. Mark

Mark a section of the experiment at air saturation, when signal stability is reached. This should be done in real-time to save default calibration information. Corrections are possible after disconnecting from the O2k. For calibration, follow steps (1) to (5):

Select a graph by a click (left mouse button) into the graph or directly by step 2.
Select the oxygen signal as the active plot by a click on Y1 in the figure legend on the right of the graph. The active plot is highlighted.
Only if Mouse Control: Zoom mode has been activated: Select Mouse Control: Mark in the Graph menu or press [Ctrl+M].
Set a mark: Hold [Shift] and click the left mouse button, move the cursor along the time axis, release the left mouse button at the end of the section to be marked. Remove a section of the mark or the total mark: Move the cursor with [Shift+right-click] along the time axis, release the mouse button at the end of the section of the mark to be deleted.
Rename the mark: Left mouse click on the bar of the mark. Rename the mark for air calibration as “R1” (and the mark for zero calibration as “R0”).

3. Calibrate

Calibration window

Open the DatLab calibration window by double clicking on O2 calib. for O2k-chamber A or B in the O2k-status line (bottom left and right). Alternatively, press [F5] to open the calibration window for the active plot.
Left click on the pull-down button and select the appropriate mark R1. The average voltage (Raw signal [V]) recorded over the marked section is shown in the corresponding field on the right. The corresponding signal stability is displayed as the uncorrected negative slope of the signal during calibration in [pmol∙s⁻¹∙mL⁻¹]. Temperature and barometric pressure are displayed as measured over the marked section. Calibration values R1 and R0 can be edited numerically, without exerting an influence on c1. If the temperature or barometric pressure are edited, then c1 is recalculated for the changed conditions.
If not set previously, enter the oxygen solubility factor of the medium, F_M, relative to pure water. For more info on this factor: MiPNet06.03 POS-calibration-SOP. For documentation purposes, enter the name of the experimental incubation medium in the corresponding field.
Left click on Calibrate and copy to clipboard. After clicking on this button all changes in the entire calibration window are applied. The entire plot of oxygen concentration is re-calibrated [µM = nmol/mL], and the corresponding negative slope or volume-specific oxygen flux [pmol∙s⁻¹∙mL⁻¹] is now based on this new calibration. Click on Calibrate and copy to clipboard before closing the calibration window. Thereby, calibration is activated and calibration parameters are automatically copied to clipboard for entry into the spreadsheet "Template O2 calibration.xlsx".

Zero calibration

1. Mark

Mark a section of the experiment after full depletion of oxygen, when signal stability is reached. This should be done in real-time to save default calibration information. Corrections are possible after disconnecting from the O2k. For calibration, follow steps (1) to (5):

Select a graph by a click (left mouse button) into the graph or directly by step 2.
Select the oxygen signal as the active plot by a click on Y1 in the figure legend on the right of the graph. The active plot is highlighted.
Only if Mouse Control: Zoom mode has been activated: Select Mouse Control: Mark in the Graph menu or press [Ctrl+M].
Set a mark: Hold [Shift] and click the left mouse button, move the cursor along the time axis, release the left mouse button at the end of the section to be marked. Remove a section of the mark or the total mark: Move the cursor with [Shift+right-click] along the time axis, release the mouse button at the end of the section of the mark to be deleted.
Rename the mark: Left mouse click on the bar of the mark. Rename the mark for zero calibration as “R0”.

2. Calibrate

Open the DatLab calibration window by double clicking on O2 calib. for O2k-chamber A or B in the O2k-status line (bottom left and right). Alternatively, press [F5] to open the calibration window for the active plot.
Left click on the pull-down button and select the appropriate mark R0. Many times the zero calibration value is used from a previous experiment. The displayed temperature and pressure are without influence on the calibration calculations for zero oxygen. The stable zero signal, R0, should be <2 % of the signal at air saturation, but <5 % is acceptable. Most importantly, the zero signal must be stable. The average voltage (Raw signal [V]) recorded over the marked section is shown in the corresponding field on the right. The corresponding signal stability is displayed as the uncorrected negative slope of the signal during calibration in [pmol∙s⁻¹∙mL⁻¹]. Temperature and barometric pressure are displayed as measured over the marked section. Calibration values R1 and R0 can be edited numerically, without exerting an influence on c1. If the temperature or barometric pressure are edited, then c1 is recalculated for the changed conditions.
If not set previously, enter the oxygen solubility factor of the medium, F_M, relative to pure water. For more info on this factor: MiPNet06.03 POS-calibration-SOP. For documentation purposes, enter the name of the experimental incubation medium in the corresponding field.
Left click on Calibrate and copy to clipboard. After clicking on this button all changes in the entire calibration window are applied. The entire plot of oxygen concentration is re-calibrated [µM = nmol/mL], and the corresponding negative slope or volume-specific oxygen flux [pmol∙s⁻¹∙mL⁻¹] is now based on this new calibration. Calibration parameters are automatically copied to clipboard for entry into the spreadsheet "Template O2 calibration.xlsx".

Oxygen calibration in instrumental and experimental DatLab files

Instrumental calibration

It is common practice to save instrumental calibration tests in separate DatLab files, which include (1) air calibration and zero oxygen calibration in the same calibration file, or (2) air calibration and zero oxygen calibration in different calibration files. Step 1: Select calibration sections from Marks (R1, R0) while DatLab is connected to the O2k during an instrumental calibration run. Step 2: Click on Calibrate and copy to clipboard in the O2 calibration window (in menu 'Calibration' > 'Oxygen, O2'). Step 3: Click on Save and disconnect in menu File. The calibration file is saved, and DatLab disconnects from the O2k.

Calibration parameters are automatically transferred to experimental DatLab files

Step 4: When a new DatLab file is recorded any time later, the latest calibration parameters are automatically applied in the new DatLab file — independent of maintaining the O2k in operation or switching it off, and independent of maintaining DatLab active or re-opening it after exit.

A calibration can be performed and calibration parameters saved during an experimental run

In the specific case of an experiment that involves an aerobic-anoxic transition, zero oxygen calibration is performed within the experimental file, setting a Mark (R0) on the oxygen signal in the anoxic section, and clicking on Calibrate and copy to clipboard in the O₂ calibration window (in menu 'Calibration' > 'Oxygen, O2').

Calibration parameters can be selectively imported from any DatLab file

Step 5: In addition, the calibration parameter for air calibration, zero oxygen calibration, or both can be imported from a selected DatLab file. This is particularly important, if a zero calibration is performed after completion and saving an experimental file. Step 6: A single calibration value (R1 or R0) can be edited manually. Take Step 7 to apply the selected calibration parameters.

Real-time vs. disconnected

DatLab uses calibration values applied in real-time (connected to the O2k, recording data) as default values for future experiments. When calibration values are edited in the disconnected mode, they apply only to the current file and will not be used as a new default in experiments. This allows to re-calibrate old files without overwriting the current default values for calibration. Ideally, calibration values that should be used as new defaults are applied in real-time when the experiment is still running. If the DatLab-calibration is performed after disconnecting, these calibration parameters can be read into other DatLab files using the "Copy from file" function and "Calibrate and copy to clipboard".

Before disconnecting the O2k from DatLab, calibration information is automatically saved and available upon re-connecting the O2k, even if you exit DatLab and start the program again. The current calibration parameters are displayed when opening the O₂ calibration window [F5].

O₂ calibration and Quality Control

Quality control - DatLab 8

Quality control - DatLab 7

Oxygen sensor test

The oxygen sensor test starts in an open chamber.

1. Before final equilibration, perform a stirrer test, switching both stirrers automatically off for 30 s.

Stirrer test for quality control (standard 30 s) with 30 min time scale displayed with Graph Layout “02-Calibration - Background” (MiR05; 37 °C; data recording interval: 2 s; slope smoothing: 40 data points).

2. About 20 minutes are required for air equilibration after temperature equilibration of the incubation medium, visualized as stabilization of the Peltier power (Fig. Quality control; time scale is 01:10 hh:mm).

Quality control label a: Upon automatic re-start of the stirrer (On), the increase of the oxygen signal should be rapid and monoexponential.

Quality control label b: The raw signal (blue plot; 1 V = 1 µA at Gain 1) should be 1 to 3 V at 25 to 37 °C at sea level up to 1000 m (p_b 101 to 90 kPa). At a Gain setting of 2 the raw signal [V] is multiplied by 2.

3. Within 40 minutes, the oxygen signals should be stable with an O2 slope (uncorrected) close to zero.

Quality control label c: Signal noise should be low, reflected in a noise of the O2 slope (red plot) within ± 2 (± 4 is acceptable) pmol∙s⁻¹∙mL⁻¹ at a data recording interval of 2 s and 40 data points selected for calculation of the slope.

4. Set a mark on the oxygen signal (R1) and open the DatLab O2 calibration window (MiPNet06.03 POS-calibration-SOP).

Quality control label d: The slope uncorrected should be within ± 1 pmol∙s⁻¹∙mL⁻¹ averaged across the section of the experiment marked as R1 for air calibration (d). The recorded POS signal should be close to the previous calibration under identical experimental conditions (O2 Calibration window, label b’).

5. Close the chamber and if required, perform a zero oxygen calibration.

Quality control label e: After closing the chamber, select plot Y2 and set mark J°1. O₂ slope neg. should be within 3.0 ± 1 pmol∙s⁻¹∙mL⁻¹.

O₂ slope neg. values higher than 4.0 pmol∙s⁻¹∙mL⁻¹ indicate:

» Biological contamination.

» Air bubbles in the Closed chamber: switch on the illumination of the O2k and inspect the O2k-Chamber through the front window. Remove any air bubbles.

» A large volume of medium collected in the receptacle of the stopper: siphon off excess medium.

» A larger chamber volume: check O2k-Chamber volume calibration.

Quality control label f: The zero signal at mark R0 for zero calibration should be <2 % of R1 (stable at <5 % is acceptable).

O₂-sensor test: when?

An O₂-sensor test should be performed:

After switching on the O2k, every day: air calibration, stirrer test, quality control (Calibration and quality control).
Zero oxygen calibration: from time to time over weeks; bracketing zero oxygen calibrations when working at low oxygen (Zero calibration).
After application of a new membrane and O₂-sensor service: in some cases, the signal of the OroboPOS improves (higher signal stability, less noise, shorter response time) when the O2k remains switched on overnight (O2k-Chambers filled with 70 % EtOH or H₂O if the traces will be evaluated in the morning).
For O2k-Quality Control (O2k-QC) of instrumental performance.
Before an O2k-chamber test (Instrumental O2 background test).
During troubleshooting procedures, when switching components between the two chambers, a quick sensor test is performed after each step (stirrer test, sensor signal).

Dynamic POS calibration = Calibration of the response time of the POS.
Static POS calibration = Two-point calibration of the polarographic oxygen sensor.

Instrumental setup

Stopper-spacers used for air calibration with white PVDF stopper (Chamber A) or black PEEK stopper (Chamber B).

Frequently, assumed hardware problems turn out to be a simple calibration problem.

Air saturation is achieved by stirring the aqueous medium in contact with air in the O2k-chamber without sample.

Add incubation medium into the chambers with an excess volume of at least 0.1 mL above the experimental chamber volume (2 mL or 0.5 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 the addition of the medium.
Insert the stoppers slowly to their volume-calibrated position. Suck off excess medium ejected through the injection capillary and remaining 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 - Open chamber position. 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 p_O2 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 to the right). A stirrer test [F9] is performed early 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).

Zero oxygen calibration may be achieved by chemical or biological depletion of oxygen. (1) Chemical: Following chamber assembly or exchange of the POS membrane, zero solution (Na-dithionite, OroboPOS-Service Kit) is titrated into the chamber, which is part of the automatic TIP2k-supported instrumental background test (MiPNet14.06). (2) Biological: Zero calibration can be performed with mitochondria or cell suspensions that allow complete oxygen depletion.

Air calibration: Oxygen concentration (blue plot; 180 µM; scale from 0 to 250 nmol/mL or 0 to 250 µM) over 1 h after switching on the O2k (Power-O2k P4, chamber A) and setting the experimental temperature at 37 °C, using medium stirred for equilibration with a gas phase of air at 575 m altitude. The red plot is the negative slope of oxygen concentration over time [pmol∙s⁻¹∙mL⁻¹] on the right Y-axis, with zero in the middle position. A slope of zero (for uncorrected ‘O₂ Slope neg.’) indicates a constant O₂ signal over time. (2014-07-24 P4-01.DLD)

Troubleshooting

* Customer ID: Erzsebet Polyak, University of Wisconsin at Madison

Question 1: We have different oxygen levels in chamber A and B.

Question 2: In chamber A after adding dithionite the signal does not go down below 100 µM.

Answer 1: Different initial oxygen levels after switching on the instrument are due to missing calibration. Sensors need to be air calibrated on each experimental day to account for changes in barometric pressure or to accommodate changes in experimental temperature or medium.

Answer 2: The zero voltage in chamber A is at 0.8996 V, which is too high. Zero voltage should not exceed 5 % (usually lower than 2 %) of the voltage obtained at air saturation (R1). You should perform a complete POS service.