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 automatic calibration routine by DatLab.^
MitoPedia O2k and high-resolution respirometry:O2k-Open Support
- 1 Description
- 2 Air calibration - general description
- 3 Air calibration in DatLab
- 4 Zero calibration - general description
- 5 Zero calibration in DatLab
- 6 DatLab calibration: real-time vs. disconnected
- 7 Calibration and quality control
- 8 O2-sensor test: when?
- 9 Instrumental background oxygen flux - general
- 10 Instrumental background oxygen flux in DatLab
- 11 Static and dynamic calibration
- 12 O2k-Open Support cases
Air calibration - general description
- 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 - 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 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 to the right). 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).
Air calibration in DatLab
- 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 lines, left Y-axis) and O2 slope (not corrected for instrumental background; red lines, right Y-axis) are displayed on the top graph for the left chamber, and below for the right 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 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
- 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.mL)]. 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 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, FM, 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 coresponding 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.mL)] is now based on this new calibration. Calibration parameters are automatically copied to clipboard for entry into the spreadsheet O2 calibration.xlsx.
Zero calibration - general description
- Zero oxygen calibration may be achieved by different means. Routinely, 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).
- Alternatively, zero calibration can be done with mitochondria or cell suspensions that allow complete oxygen depletion.
Zero calibration in DatLab
- 1. Mark
- Mark a section of the experiment after full depletion of oxygen, when signal stability is reached. This should be done 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.
- Rename the mark: Left mouse click on the bar of the mark. Rename the mark for air 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.ml)]. 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 temperature or barometric pressure are edited, then c1 is recalculated for the changed conditions.
- 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.ml)] is now based on this new calibration. Calibration parameters are automatically copied to clipboard for entry into the spreadsheet O2 calibration.xlsx.
DatLab calibration: real-time vs. disconnected
- DatLab uses calibration values applied 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 real-time when the experiment is still running. However, 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 Oxygraph-2k from DatLab, calibration information is automatically saved and available upon connecting the Oxygraph-2k, even if you exit DatLab and start the program again. The current calibration parameters are displayed when opening the O2 calibration window F5.
Calibration and quality control
- The POS sensor test
- About 20 min are required for approximate air equilibration after temperature equilibration of the incubation medium.
- Even before final equilibration, perform a stirrer test [F9], switching both stirrers automatically off for 30 s.
- Quality control a: Upon automatic re-start of the stirrer (On), the increase of the oxygen signal should be rapid and monoexponential (see Fig. right, label )
- Quality control b: The raw signal (blue plot; 1 V = 1 µA at gain 1) should be close to 1 to 3 V at 25 to 37 °C at sea level up to 1000 m (pb 101 to 90 kPa). At gain setting of 2 the raw signal [V] is multiplied by 2.
- Set a mark on the oxygen signal (R1) and click on O2 Calib. to open the DatLab O2 calibration. Quality control label : The slope uncorrected should be within ±1 pmol/s∙ml) 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. See O2-Calibration window (see Fig. right, label label ).
- Continue with an instrumental O2 background test and perform a zero oxygen calibration. Quality control label label : 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 over night (O2k-Chambers filled with 70% EtOH).
- 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).
Instrumental background oxygen flux - general
- Instrumental background oxygen flux derives from oxygen fluxes into or out of the respirometer chamber that are not directly related to respiratory activity of the sample. These fluxes are caused by backdiffusion into the chamber at low oxygen pressure, oxygen diffusion out of the chamber at elevated oxygen levels, and oxygen consumption by the polarographic oxygen sensor (OroboPOS). Correction for instrumental background oxygen flux is a standard in high-resolution respirometry, automatically performed by DatLab. Background measurements provide a quality control of instrument function. In the Oroboros O2k, background corrections are usually within a few % of experimental flux over the entire experimental oxygen range. At minimum activities, however, even the small background effects become significant and require compliance to standard operating procedures described here.
Instrumental background oxygen flux in DatLab
- Instrumental O2 background tests can be perfomed using the TIP2k or with manual injections. Detailed instructions are provided here: MiPNet14.06_Instrumental_O2_background
Static and dynamic calibration
- Static POS calibration = Two-point calibration of the polarographic oxygen sensor.
- DatLab converts the raw signal to oxygen concentrations by applying conversion factors determined during a POS calibration comprising an air calibration and a zero calibration. For each gain a different set of calibration values is necessary. Frequently, assumed hardware problems turn out to be a simple calibration problem. This can easily be detected by observing the Raw signal.
- Dynamic POS calibration = Calibration of the response time of the POS.
O2k-Open Support cases
Q1: We have different oxygen levels in chamber A and B.
Q2: In chamber A after adding dithionite the signal does not go down below 100µM.
A1: 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 accomodate changes in experimental temperature or medium.
A2: The zero voltage in chamber A is at 0.8996V, which is too high. Zero voltage should not exceed 10% (usually is lower than 5%) of that seen in R1. You should do a full POS service.
Labels: MiParea: Instruments;methods
HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol
DatLab, DL7, DL6a7
MitoPedia methods: Respirometry
MitoPedia O2k and high-resolution respirometry: DatLab