Difference between revisions of "MitoPedia: DatLab"

From Bioblast
Jump to: navigation, search
 
Line 18: Line 18:
 
::::» [[Close - DatLab| Close]]
 
::::» [[Close - DatLab| Close]]
 
::::» [[Save - DatLab | Save]]
 
::::» [[Save - DatLab | Save]]
::::» [[Save and Disconnect]]
+
::::» [[Save and Disconnect | Save and disconnect]]
 
::::» [[Save as - DatLab | Save as]]
 
::::» [[Save as - DatLab | Save as]]
 
::::» [[File search - DatLab| File search]]
 
::::» [[File search - DatLab| File search]]

Latest revision as of 09:08, 13 September 2019

Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         Oroboros O2k         MiP and biochemistry         Preprints and history



MitoPedia

MitoPedia: DatLab

MitoPedia - high-resolution terminology - matching measurements at high-resolution.
The MitoPedia terminology is developed continuously in the spirit of Gentle Science.

Oroboros support
Welcome at MitoPedia: DatLab. Here you find Oroboros support for DatLab and the O2k.


List of DatLab menues
Template NextGen-O2k.jpg

DatLab-bar.jpg

DatLab
DatLab installation
» MiPNet21.16 DatLab 7 Innovations
File-menu.png

File

» Open
» Close
» Save
» Save and disconnect
» Save as
» File search
» Delete
» Import DatLab templates DatLab templates
» Export
» Change user: User
» Manage users: User
O2k-menu.png

Oroboros O2k

» O2k configuration
» O2k channel labels
» O2k control
» Stirrer A on/off
» Stirrer B on/off
» Stirrer test
» Illumination on/off
» Manage setups: Manage setups and templates
TIP2k-menu.png

TIP2k

» TIP2k control: Titration-Injection microPump
» Manage setups: Manage setups and templates
Protocol-menu.png

Protocol

» Run DL-Protocol/Set O2 limit
» Instrumental: Browse DL-Protocols and templates
» SUIT: Browse DL-Protocols and templates
» Install Oroboros protocol package
» Browse www.bioblast.at/index.php./MitoPedia: SUIT
» Enable DL-Protocol editing
Experiment-menu.png

Experiment

» Sample: Sample
» Exp. log: Experimental log
» Add event: Events
Calibration-menu.png

Calibration

» O2: O2 calibration
» Amp: Amp calibration
» pX: pX calibration
Flux-Slope-menu.png

Flux / Slope

» O2: Flux / Slope
» Amp: Flux / Slope
» pX: Flux / Slope
» Proton Flux / Slope
Graph-menu.png

Graph

» Add graph/Delete bottom graph: Add/Delete
» Select plots: Select plots - DatLab
» Scaling: Scaling - DatLab
» Info: Graph / Info
» Synchronous time axes
» Autoscale
» Mouse control: Mark and Zoom
» Full screen
» Display numerical value
» Display Power-O2k
» Options: Graph options
» Copy to clipboard


Layout-menu.png

Layout

» Info / Load / Save: Layout: Info/ Load/ Safe
» Standard layouts - O2k-FluoRespirometer: Standard layouts - O2k-FluoRespirometer
» Lab layouts: Lab layouts
» User: Name: User: Name
Marks-menu.png

Marks

» Edit mark information: Marks
» Statistics: Mark statistics
» Manage mark stat. templates: Manage setups and templates
» Specifications: Mark specifications
» Manage mark specifications templates: Manage setups and templates
» Copy marks


DatLab glossary

TermAbbreviationDescription
Add Graph/Delete bottom graphThe active graph is selected by a left click into the graph. The active graph is highlighted and indicated by the Oroboros logo.

Add: A new graph is added at the bottom of the screen. Select plots for display in the new graph, Ctrl+F6

Delete: By clicking Delete bottom graph in the Graph-menu in DatLab, the bottom graph is deleted, which reappears with the same layout by Add.
Advancement per volumedtrY [MU∙L-1]Advancement per volume or volume-specific advancement, dtrY, is related to advancement of a transformation, dtrY = dtrξV-1 [MU∙L-1]. Compare dtrY with the amount of substance j per volume, cj (concentration), related to amount, cj = njV-1 [mol∙V-1]. Advancement per volume is particularly introduced for chemical reactions, drY, and has the dimension of concentration (amount per volume [mol∙L-1]). In an open system at steady-state, however, the concentration does not change as the reaction advances. Only in closed systems and isolated systems, specific advancement equals the change in concentration divided by the stoichiometric number,
drY = dcj/νj (closed system) 
drY = drcj/νj (general) 

With a focus on internal transformations (i; specifically: chemical reactions, r), dcj is replaced by the partial change of concentration, drcj (a transformation variable or process variable). drcj contributes to the total change of concentration, dcj (a system variable or variable of state). In open systems at steady-state, drcj is compensated by external processes, decj = -drcj, exerting an effect on the total concentration change of substance j,

dcj = drcj + decj = 0 (steady state)
dcj = drcj + decj (general)
Air calibrationR1Air calibration of an oxygen sensor (polarographic oxygen sensor) is performed routinely on any day before starting a respirometric experiment. The volume fraction of oxygen in dry air is constant. An aqueous solution in equilibrium with air has the same partial pressure as that in water vapour saturated air. The water vapour is a function of temperature only. The partial oxygen pressure in aqueous solution in equilibrium with air is, therefore, a function of total barometric pressure and temperature. Bubbling an aqueous solution with air generates deviations from barometric pressure within small gas bubbles and is, therefore, not recommended. To equilibrate an aqueous solution ata known partial pressure of oxygen [kPa], the aqueous solution is stirred rigorously in a chamber enclosing air at constant temperature. The concentration of oxygen, cO2 [µM], is obtained at any partial pressure by multiplying the partial pressure by the oxygen solubility, SO2 [µM/kPa]. SO2 is a function of temperature and composition of the salt solution, and is thus a function of the experimental medium. The solubility factor of the medium, FM, expresses the oxygen solubility relative to pure water at any experimental temperature. FM is 0.89 in serum (37 °C) and 0.92 in MiR06 or MiR05 (30 °C and 37 °C).
Amp calibration - DatLabAmp calibration indicates the calibration of the amperometric O2k-channel.
Automatic pan - DatLabAutomatic pan (only for real-time data recording) toggles automatic panning on/off by clicking in the O2k status line. If it is on (green), the time range is maintained while the time axis always shows the currently recorded data, i.e. the value of the offset (minimum value) increases as experimental time proceeds. If it is off (yellow), the time axis is static. This allows for manually panning backwards to observe previous sections of the experiment at a given time range. In this mode, the actual experimental time may be off-scale. Toggle between "Pan auto" and "Pan off" by a left-click on the text. It does not influence continuous data recording. It is recommended to maintain automatic panning on during the experiment, except for specifically viewing earlier sections of the experiment.
AutoscaleAutoscale zooms in or out of the selected period with Autoscale time axis, Autoscale Y1 (Y2) axes and Automatic pan.
Autoscale Y1 (Y2) axesAutoscale Y1 (Y2) axes: Autoscaling the measured values (full data range) on the Y1 (Y2) axis in the selected plot.
Autoscale time axisAutoscale time axis gives an overview of the entire experimental period.
Barometric pressurepb [Pa]Barometric pressure, pb, is an important variable measured for calibration of oxygen sensors in solutions equilibrated with air. The atm-standard pressure (1 atm = 760 mmHg = 101.325 kPa) has been replaced by the SI standard pressure of 100 kPa. The partial pressure of oxygen, pO2, in air is a function of barometric pressure, which changes with altitude and locally with weather conditions. The partial oxygen pressure declines by 12% to 14% per 1,000 m up to 6,000 m altitude, and by 15% to 17% per 1,000 m between 6,000 and 9,000 m altitude. The O2k-Barometric Pressure Transducer is built into the Oroboros O2k as a basis for accurate air calibrations in high-resolution respirometry. For highest-level accuracy of calculation of oxygen pressure, it is recommended to compare at regular intervals the barometric pressure recording provided by the O2k with a calibrated barometric pressure recording at an identical time point and identical altitude. The concept of gas pressure or barometric pressure can be related to the generalized concept of isomorphic pressure.
Block temperatureThe block temperature of the Oroboros O2k is the continuously measured temperature of the copper block, housing the two glass chambers of the O2k. The block temperature is recorded by DatLab as one of the O2k system channels.
Chamber volumeThe standard Chamber volume of the O2k is 2.0 ml of aqueous medium with or without sample, excluding the volume of the stirrer and the volume of the capillary of the stopper.
ChannelF7» See O2k signals and output
Chemical background correction of oxygen fluxChemical background correction of oxygen flux is the correction of oxygen flux for the side reaction of autooxidation, as a function of oxygen concentration.
Close - DatLabCtrl+F4Close a DLD file. A window "Save changes to file?" pops up offering the options to close the file after saving the changes, or close the file without saving any modifications on the presently open file.
Connect to O2kConnect to O2k connects DatLab with the O2k. Select the USB port (or Serial port) with the corresponding cable connecting your PC to the O2k. Select the subdirectory for saving the DLD file. Then data recording starts with experimental time set at zero.
Connection windowAfter starting DatLab either the Connection window opens automatically by default or open O2k control by pressing [F7] and select the communication port.
Copy marksCopy marks from a selected plot to the active plot
Copy marks fromCopy marks from: Marks in DatLab are copied from a seleted Plot to the active plot.
Copy to clipboardIn DatLab Copy to clipboard can be used to copy selected graphs or values and to paste them to your preferred program or file (e.g. Word, Excel).
Custom labelA Custom label can be entered in this box to rename the axis label. Two lines are available for the axis name and unit.
DatLab 2DL2DatLab 2 is a MS-DOS programme. It may be necessary to use an emulator to be able to run DatLab 2 on modern computers, especially for the DL2 graphic window (available free of charge):
  • Windows 7 + DOSBOX
  • Linux + DOSemu
  • FreeDOS: www.freedos.org (will also reduce the available CPU power if necessary)
  • "CPUkiller" - to reduce available CPU power.
DatLab data fileDLDThe file type generated by DatLab is *.DLD.
DatLab error messagesCommon DatLab error messages and according actions and solutions are listed here.
DatLab installation
DatLab
We recommend a 'clean install' for DatLab installation: rename your previous DatLab programme subdirectory (e.g. C:\DatLab_OLD). The standard Instrumental and SUIT DL-Protocols package is automatically implemented with the simple DatLab programme installation.
DatLab oxygen flux: performance and data analysisThe quality of the results are strongly affected by the performance and data analysis. Therefore, we provide guidelines for performing and evaluating respirometric assays.
DatLab templatesDatLab templates can be imported for O2k-setups, graph layouts, mark names, TIP2k setups and marks statistics configurations. To do so, go to DatLab menu 'File\Import\DatLab templates' and choose an apporpriate DatLab template file (*.DLT) from the filesystem. The standard template file generated during DatLab installation can be found in the folder DatLab\DLTemplates.
See also » Manage setups and templates
DatLab-Analysis templatesGo in DatLab to Mark statistics (F2), select which type of marks you want to export ("All marks in plot" or "DL-Protocol marks", with 3 possibilities each), then click on [Copy to clipboard] to copy selected values and paste them to a DatLab-Analysis template for numerical and graphical data analysis.
Data recording intervalF7The data recording interval is the time interval at which data is sampled with an instrument. In DatLab the data recording interval is set in the O2k control window. The system default value is 2 s. A lower data recording interval is selected for kinetic experiments, and when the volume-specific oxygen flux is high (>300 pmol O2·s-1·ml-1).
Technically, the O2k instrument (hardware) measures the sensor signal every 10ms (which is NOT the „data recording interval“). By the given data recording interval from DatLab (software) a discrete number of sensor signal points are taken to calculate an average value in the O2k (e.g. a data recording interval of 2 s can take 200 sensor signal points; a data recording interval of 0.5 s can take 50 sensor signal points). This average value is sent to DatLab and is recorded as a raw data point. However, there is a defined threshold: the O2k does not apply more than 200 sensor signal points to calculate the average for the raw data point. For example a data recording interval of 3 s could take 300 sensor signal points but only the 200 most recent sensor signal points are used for averaging.
Default labelThe Default label is the system default value for the axis label. These labels are changed automatically, according to the selected channel and unit. To change this label enter a Custom label.
Delete - DatLabDelete a DLD file. The decision to delete a file containing no useful data can be made most easily when viewing the traces. Only available when disconnected from the O2k.
Delete pointsSelect Delete points in the Mark information window to remove all data points in the marked section of the active plot. See also Interpolate points and Restore points or Recalculate slope.
Deselect channelsF7Channels can be selected/deselected in DatLab in the O2k configuration. Deselect all O2k-MultiSensor channels in O2k-Core applications. Select only the specifically used channels in O2k-MultiSensor applications.
Display DatLab helpDisplay DatLab help

In this section, we present some issues that could happen during your data analysis related to the graphs display and how to fix them quickly.

Case in which an issue might occur:

  • While analysing your data, trying to close the program while the graph is still being loaded. If you cancel the closing window, the graph will not be loaded at the screen.

In the event of a frozen display of the graphs, try the alternatives below:

  • Click on: Graph > Autoscale time axis
  • Click on: Graph > Scaling (F6); then press OK to confirm the scaling and induce the program to reload the graphs (no changes in the graphs are required).
Display Power-O2kThe Power-O2k number, which is set in the pull-down menu Oroboros O2k \ O2k configuration, is shown in the active graph. To show it in graphs copied to clipboard, the option "Show Oroboros icon in clipboard files" must be enabled in the Graph-menu Graph options - DatLab.
Display numerical valueIf Display numerical value the current numerical values are displayed in the graph for the active plots on the Y1 axis and Y2 axis (during data acquisition only).
Enable DL-Protocol editingin progress
Events - DatLabF4An event in DatLab is a defined point in time, labelled by a name (1 to 10 characters). An event applies to all plots of the selected O2k-Chamber. The event is shown by a vertical line in the graph and the label of the event is shown on the top (DatLab 6 and lower: on the bottom). The default name is the sequential number of the event. It is recommended to edit event labels with a minimum number of characters, and to explain the abbreviation in the 'Definition' box. The final concentration and titration volume can be entered into the corresponding boxes, if the event relates to the titration of a substance. A short comment can be entered to describe the event in detail.

Set events - Manual events are entered (real-time, connected to the O2k) by pressing [F4] at the time of the event (e.g. to indicate a manual titration into the chamber). An event belongs either to chamber A, chamber B, or both. Instrumental events are added automatically, e.g. when the stirrer (A or B) or illumination (both chambers) is switched on or off. After setting a new event the Edit event window pops up. Pressing F4 defines the time point of the event. Full attention can then be paid to the experiment. Edit the event later, as it is possible to insert an event at any chosen moment of the plotted record of the experiment by placing the cursor anywhere in the graph at the selected time point by pressing Ctrl and clicking the left mouse button. Edit event - Left click on the name of an existing event to open the Edit event window to edit or Delete event. In events obtained from a selected protocol, the entire sequence of consecutive events is defined with event names, definitions, concentrations and titration volumes. Name - Enter an event name of 1 to 10 characters. Short names (e.g. O instead of Open) are recommended. Comment - Further information can be entered into the text field. Select O2k-chamber A, B or both. The Event will be shown on plots for both or one selected chamber.

»Protocol events
Experimental codeF3An experimental code can be entered in the Sample window, containing up to 10 digits.
Experimental log - DatLabCtrl+F3Experimental log provides an automatically generated experimental protocol with detailed information about the O2k settings and calibrations, the Sample information and various Events. Time-dependent information can be viewed for a single chamber or both chambers. The filter can be selected for viewing minimum information, intermittent by default, or all information. The experimental log can be viewed and saved as a PDF file by clicking on [Preview].
Export DL-Protocol User (*.DLPU)Export DL-Protocol User (*.DLPU) Protocol possess unique D## codes and comprise a fixed sequence of events and marks which cannot be changed by the user. However, the user may edit concentrations and titration volumes of injections and store the modified protocol as user-specific DL-Protocol [File]\Export\DL-Protocol User (*.DLPU).

If users wish to alter the nature of the chemicals used or the sequence of injections, we ask them to contact support@oroboros.at.


DatLab 7.4

DatLab 7.4 offers a new feature in DL-Protocols: flexibility. Fixed sequence of events and marks can be changed (Skip/Added) in a SUIT protocol by the user. Moreover, editions of text, instructions, concentrations and titration volumes of injections in a specific DL-Protocol can be edited and saved as user-specific DL-Protocol [File]\Export\DL-Protocol User (*.DLPU).
File search - DatLabCtrl+FFile search yields a list of all files labelled by the experimental code in a selected directory . Click on the file to preview the experimental log. With File Search you can search in all folders and subfolders on your computer for DatLab files with a selected experimental code. The experimental code is entered in the DatLab file in the window "Experiment" ([F3]). When you click on a folder and press the button search, the DatLab file names will appear on the right window. Click on a DatLab file and further information (e.g. Sample information, Background information) will appear in the window below.
Flux / SlopeJFlux / Slope is the pull-down menu in DatLab for (1) normalization of flux (chamber volume-specific flux, sample-specific flux or flow, or flux control ratios), (2) flux baseline correction, (3) Instrumental background oxygen flux, and (4) flux smoothing, selection of the scaling factor, and stoichiometric normalization using a stoichiometric coefficient. A Savitzky-Golay smoothing filter is used in DatLab as a basis of calculating the time derivative (Flux / Slope) of the signal (oxygen, fluorescence, ..). For each signal channel, the signal for the measured substance X is typically calibrated as an amount of substance concentration, cX [µM = nmol/mL]. The signal of the potentiometric channel, however, is primarily expressed logarithmically as pX=-log(cX/c°) and then transformed to cX. The slope is calculated as the change of concentration over time, dcX/dt [nmol/(s · mL)]. In a chemical reaction, the change of substance X is stoichiometrically related to the changes of all other substrates and products involved in the reaction. If the stoichiometry of the reaction is normalized for substance X, then its stoichiometric coefficient is unity and νX equals 1 if the substance is a product formed in the reaction, but νX equals -1 if the substance is a substrate consumed in the reaction. Oxygen is formed in photosynthesis and νX=1 when expressing photosynthesis as oxygen flux. Oxygen is consumed in aerobic respiration and νX=-1 when expressing respiration as oxygen flux.
Flux analysis - DatLabThe strategy of Flux analysis using DatLab depends on the research question and the corresponding settings applied in DatLab when recording the data with the O2k. Usng SUIT protocols, a sequence of respiratory steady-states is measured, marks are set, and numerical data are summarized in Mark statistics (F2). An AI approach is kept in mind when describing guidelines for evaluation of steady-states during data recording and analysis.
Flux baseline correctionbcFlux baseline correction provides the option to display the plot and all values of the flux (or flow, or flux control ratio) as the total flux, J, minus a baseline flux, J0.
JV(bc) = JV - JV0
JV = (dc/dt) · ν-1 · SF - V
For the oxygen channel, JV is O2 flux per volume [pmol/(s·ml)] (or volume-specific O2 flux), c is the oxygen conentration [nmol/ml = µmol/l = µM], dc/dt is the (positive) slope of oxygen concentration over time [nmol/(s · ml)], ν-1 = -1 is the stoichiometric coefficient for the reaction of oxygen consumption (oxygen is removed in the chemical reaction, thus the stoichiometric coefficient is negative, expressing oxygen flux as the negative slope), SF=1,000 is the scaling factor (converting units for the amount of oxygen from nmol to pmol), and V is the volume-specific background oxygen flux (Instrumental background oxygen flux). Further details: Flux / Slope.
Flux control ratioFCRFlux control ratios (FCR), are ratios of oxygen flux in different respiratory control states, normalized for maximum flux in a common reference state, to obtain theoretical lower and upper limits of 0.0 and 1.0 (0% and 100%). For a given protocol or set of respiratory protocols, flux control ratios provide a fingerprint of coupling and substrate control independent of (i) mt-content in cells or tissues, (ii) purification in preparations of isolated mitochondria, and (iii) assay conditions for determination of tissue mass or mt-markers external to a respiratory protocol (CS, protein, stereology, etc.). FCR obtained from a single respirometric incubation with sequential titrations (sequential protocol; SUIT protocol) provide an internal normalization, expressing respiratory control independent of mitochondrial content and thus independent of a marker for mitochondrial amount. FCR obtained from separate (parallel) protocols depend on equal distribution of subsamples obtained from a homogenous mt-preparation or determination of a common mitochondrial marker.
Full screenBy clicking/enabling Full screen in the Graph-menu in DatLab the currently selected graph is shown alone on the full screen (On) or together with the other defined graphs (Off). Full screen is particularly useful for a single channel overview and for Copy to clipboard [ALT+G B].
Graph layout - DatLab» See Layout for DatLab graphs.
Graph options - DatLabSeveral display options can be applied to a DatLab graph under Graph options.
IlluminationF10The chambers of the Oroboros O2k are illuminated by an internal LED. The illumination is switched on and off in DatLab during the experiment by pressing [F10]. This illumination must be distinguished from light introduced into the chambers by LEDs for the purpose of spectrophotometric and fluorometric measurements. For these, the internal illumination must be switched off.
Illumination on/offF10The illumination in both chambers is switched on/off.
Install Oroboros protocol packageThe standard Instrumental and SUIT DL-Protocols package is automatically implemented with the simple DatLab programme installation. We recommend a 'clean install': rename your previous DatLab programme subdirectory (e.g. C:\DatLab_OLD). Updates and newly developed DL protocols can be simply downloaded by clicking on [Protocols]\Install Oroboros protocol package.
Instrumental: Browse DL-Protocols and templatesDL-Protocols for instrumental calibration and maintenance can be browed from inside DatLab 7.4, example traces with instructions and brief explanatory texts and templates for data evaluation can be opened. Click on menu [Protocols]\Instrumental: Browse DL-Protocols and templates to open a folder with all the DatLab protocols (DLP) for cleaning, calibration, and background determination provided with the USB. Select a sub-directory and open an example trace and/or template as desired.
Interpolate pointsSelect Interpolate points in the Mark information window to interpolate all data points in the marked section of the active graph. See also Delete points and Restore points or Recalculate slope.
Laboratory titration sheetLaboratory titration sheet contains the sequential titrations in a specific Substrate-uncoupler-inhibitor titration (SUIT) protocol. The laboratory titration sheets for different SUIT protocols are incorporated in DatLab (DL7.1): Protocols in DatLab
Layout for DatLab graphsA Layout in DatLab from the Layout menu or the Graph menu is a predefined selection for standardized display of graphs, plots to be displayed at specific scalings. Any defined graph layout can be selected from the Layout menu for defining initial settings for the plots [Ctrl+F6] and scaling [F6]. A layout can be modified and saved under a specific name.
Manage setups and templates - DatLabSetups and templates in DatLab can be renamed or deleted under Manage setups or Manage templates.
Mark informationMarks» See Marks - DatLab
Mark specifications - DatLabMark specifications in DatLab allow the user to rename Marks in the active plot and save/recall the settings. Rename marks individually by clicking into the horizontal bar, or use corresponding templates for renaming the entire sequence of marks.
Mark statistics - DatLabF2In Mark statistics one Plot is selected as a source for Marks over sections of time. Values (e.g. medians) are displayed for these time sections of the source plot and of all selected plots.
Marks - DatLabMarks in DatLab define sections of a plot recorded over time. Marks are set by the user in real-time, or post-experimentally for basic level data analysis. Set Marks to obtain the median, average, standard deviation, outlier index and range of the data within the mark, for calibration of the oxygen signal, flux analysis, or to delete marked data points. Marks are shown by a horizontal bar in the active plot. The default Mark names are given automatically in numerical sequence, independent for each plot. Rename marks individually by clicking into the horizontal bar, or use corresponding templates for renaming the entire sequence of marks.Several marks can be set on any plot, but marks cannot overlap within a plot and are separated by one or more data points which are not marked.
Mouse control: MarkCtrl+MThe mark mode is active by default, can be selected in the menu or by [Ctrl+M]. If Mouse control: Mark is enabled, specific sections of the experiment can be marked in each plot.

Usually, marks are set on the plot for oxygen concentration for calibration, whereas marks on the plot for oxygen flux are set for exporting the median or average of flux to a table.

»More details: Marks - DatLab.
Mouse control: ZoomCtrl+ZSelect Mouse Control: Zoom in the Graph-menu or press [Ctrl+Z].
O2k chamber volume calibrationThe O2k chamber volume calibration has to be done before getting started with the Oroboros O2k to guarantee a standard chamber volume of 2.0 mL.
O2k channel labelsDefault channel labels can now be changed, and new labels set by the user. E.g., rename the Amperometric channel, Amp, to 'H2O2' for H2O2 measurements by fluorometry; rename the potentiometric channel, pX, to TPP+ for mitochondrial membrane measurements with the O2k-pH ISE-Module. For changing the label, go to menu [Oroboros O2k]\O2k channel labels and set the new channel label as desired.
O2k configurationIn O2k configuration, channels (amperometric and potentiometric) can be switched on/off by selecting the according tick box. The Power-O2k number (P1, P2, ..) and numbers for O2 sensors, Amp sensors, pX electrodes and pX reference electrodes are entered or edited here. With the O2k-FluoRespirometer (O2k-Series H), the serial numbers of the Smart Fluo-Sensors are shown automatically under [Amperometric, Amp]. The O2k configuration window pops up when DatLab starts and "Connect to O2k" is pressed for the first time. It is also accessible from the menu "Oroboros O2k" and from within the O2k control and Mark statistics windows.
O2k controlF7After selection of an O2k setup in the O2k control [F7] window, followed by a left-click Send to O2k, only the following control functions are routinely required during experimental operations.
O2k serial numberThe O2k serial number (hardware number) of the Oroboros O2k is printed on the sticker on the rear of the O2k housing, starting with a capital letter (O2k series) followed by four numbers.
O2k seriesThe O2k series is specified as the first character of the O2k serial number of the Oroboros O2k, printed on the sticker on the rear of the O2k housing. A serial number A-#### or B-#### denotes an Oxygraph from the A or B series, while C-#### denotes an Oxygraph from the C series and so on.

With DatLab running real-time connected to the O2k, the serial number of the currently connected O2k is displayed

O2k signal lineThe O2k signal line is underneath the O2k status line. It shows, depending on the O2k series, on the left side the O2k number, the time of the experiment, the oxygen raw signal of each chamber, the block temperature, the barometric pressure, the Peltier power, the recorded amperometric and potentiometric raw signal, the enviromental (room) temperature and the signal from the external temperature sensor. On the right side of the O2k signal line the current user, the DatLab version and the O2k serial number are displayed.
O2k signals and outputThree electronic channel types are available in the O2k-MultiSensor system. All channels are available twofold (dual-data), for O2k-Chambers A (left) and B (right), based on numerical signals sent at a fixed data sampling time interval (default: 2 s; range 0.2 s to >10 s).
O2k status lineO2k status line is found above the O2k signal line. It contains information about the chamber label, O2 calibration, amperometric calibration, potentiometric calibration, the block temperature, the illumination in chambers, the TIP2k status and the Automatic pan.
Open DLD fileCtrl+OWith the button Open DLD file a previously recorded DatLab file is opened. The file type is *.DLD for DatLab data files. It is not possible to open a second DLD file without closing first the currently open DatLab file. You can open simultaneously another DatLab file in a separately opened DatLab programme.
Oroboros USB-flash driveThe Oroboros USB-flash drive is delivered with the Oroboros O2k. Copy the folder "Oroboros O2k-Course on HRR" from the Oroboros USB-flash drive to your computer. This folder contains the DatLab installation program as well as tools to find topics, O2k-manuals and O2k-protocols with corresponding DatLab demo files and templates for training with DatLab.
Outlier index - DatLabOIAn outlier index (OI) is defined for DatLab analysis, derived from Pearson’s coefficient of skewness, but more specific in targeting outliers in data series recorded with the O2k. At the limit of a zero value, Y = ABS(Average + Median)/2, the OI equals Pearson’s coefficient of skewness #2 (without the multiplication factor of 3). At high Y with small standard deviation (SD), the outlier index is effectively the difference between the Average and the Median normalized for the absolute value, (Average-Median)/Y. The definition of the outlier index is,
OI = (Average-Median)/(Y + SD)
OI = (Average-Median)/[ABS(Average+Median)/2 + SD]

By comparison,

Pearson’s coefficient of skewness #2 = 3 x (Average-Median)/SD
The threshold of the absolute value of the OI is set at 0.05. If ABS(OI)>0.05 calculated for the data points within a defined Mark, the Mark window indicates the likely occurrence of outliers in the data sequence. The threshold can be set to a lab-specific or session-specific value different from the default value.
Overlay of plots - DatLabOverlay of plots from the two O2k-Chambers in a DatLab file is achieved by selecting the corresponding Graph layout. Superimposed traces of flux/flow from chambers A and B are then shown in Graph 1, and of concentration in chambers A and B in Graph 2.

There are basically two ways to superimpose traces recorded in different experiments: Export of the graphics via windows metafile or export of the data to e.g. a spreadsheet program.

If you export via wmf you also can manipulate the graphics but then usually the lines are broken up in different segments. This can be done in various programs like MS Word, Open Office Draw and even in MSPower Point, though this maybe is the worst program to do this. It is better to manipulate them in a proper program like OO Draw, convert it to an unchangeable picture and then import it to a presentation graphics. Anyway, when you import directly to Power point (or other programs), make sure not to import it as a "picture" but as a metafile. Also in some programs you might afterwards have to "break" it up, or accept a "conversion to a MS Draw object" or other similar linguistic inventions of the software gurus. For this option we suggest to do as much as possible directly in DatLab (setting colors, line widths, ..) using the options in "Plots"/"select plots" and "graph"/"options".

The “hardcore“ option is to export the data and import it into e.g. a spreadsheet program (MS Excel , OOCalc). It takes longer to have a simple overlay but gives you far less problems later and its easier to make changes later. To do this you can export your dataset "Export"/"Data to Textfile" and then go from there.
Oxygen calibration - DatLabO2 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.^
Oxygen flowIO2 [mol·s-1]Respiratory oxygen flow is the oxygen consumption per total system, which is an extensive quantity. Flow is advancement of a transformation in a system per time. Oxygen flow or respiration of a cell is distinguished from oxygen flux (e.g. per mg protein or wet weight).
Oxygen flux - instrumental backgroundJ°O2Instrumental background oxygen flux, J°O2, in a respirometer is due to oxygen consumption by the POS, and oxygen diffusion into or out of the aqueous medium in the O2k-Chamber. It is a property of the instrumental system, measured in the range of experimental oxygen levels by a standardized instrumental background test. The oxygen regime from air saturation towards zero oxygen is applied generally in experiments with isolated mitochondria and living or permeabilized cells. To overcome oxygen diffusion limitation in permeabilized fibres and homogenates, an elevated oxygen regime is applied, requiring instrumental background test in the same range of elevated oxygen.

Instrumental background correction eliminates errors by systemic flux compensation, automatically performed by DatLab. If no experimental background test has been performed, the system default values are used, which are a°=-2.0 pmol/(s·ml) for the intercept at zero oxygen concentration, and b°=0.025 for the slope of background flux as a function of oxygen concentration.

Automatic correction for the instrumental background oxygen flux is an essential standard in high resolution respirometry. At the same time an instrumental background experiment is the ultimate test for instrumental performance, evaluating chamber performance after completion of all elements of the Oxygen sensor test. The instrumental background oxygen flux measured at air saturation should reflect the theoretically predicted volume-specific oxygen consumption by the oxygen sensor. The actual agreement using experimental respiration medium provides at the same time a test that excludes microbial contamination of the medium or serves to evaluate any autoxidation processes in newly tested experimental media.
Oxygen pressurepO2 [kPa]Oxygen pressure or partial pressure of oxygen [kPa], related to oxygen concentration in solution by the oxygen solubility, SO2 [µM/kPa].
Oxygen sensor testPOS testThe O2 sensor test is an important component of the MitoFit Quality Control System. The OroboPOS sensor test is described in detail in MiPNet06.03 POS-calibration-SOP, is performed after switching on the Oroboros O2k, and is required as a basis of technical service of the instrument.
Oxygen signalThe oxygen signal of the Oroboros O2k is transmitted from the electrochemical polarographic oxygen sensor (OroboPOS) for each of the two O2k chambers to DatLab. The primary signal is a current [mAmp] which is converted into a voltage [V] (raw signal), and calibrated in SI units for amount of substrance concentration [µmol·L-1 or µM].
Oxygen solubilitySO2 [µM/kPa]The oxygen solubility, SO2 [µM/kPa], expresses the oxygen concentration in solution in equilibrium with the oxygen pressure in a gas phase, as a function of temperature and composition of the solution. SO2 is 10.56 µM/kPa in pure water at 37 °C. At standard barometric pressure (100 kPa), the oxygen concentration at air saturation is 207.3 µM at 37 °C (19.6 kPa partial oxygen pressure). In MiR06 and serum, the corresponding saturation concentrations are 191 and 184 µM. The oxygen solubility depends on temperatue and the concentrations of solutes in solution. See also: Oxygen solubility factor
Oxygen solubility factorFMThe oxygen solubility factor of the incubation medium, FM, expresses the effect of the salt concentration on oxygen solubility relative to pure water. In mitochondrial respiration medium MiR06, FM is 0.92 determined at 30 and 37 °C, and FM is 0.89 in serum at 37 °C. FM for other media may be estimated using Table 4 in MiPNet06.03. For this purpose KCl based media can be described as "seawater" of varying salinity.
PC requirementsThe PC requirements for controlling an O2k and data recording with DatLab are found here.
POS calibration - dynamicCalibration of the sensor response time. See also POS calibration - static.
POS calibration - staticF5Two-point calibration of the polarographic oxygen sensor, comprising Air calibration and Zero calibration. See also POS calibration - dynamic.
PX calibration - DatLabpX calibration Site under construction!
Plot - DatLabCtrl+F6A plot in DatLab represents a specific channel in the graph. To change the Layout for DatLab graphs go to [Graph]/Select plots to open the Graph layout window.
Polarization voltageUA polarization voltage of 600 mV to 800 mV is applied between anode and cathode of the polarographic oxygen sensor, resulting in a current when oxygen is consumed. The current is converted by the electronics to a voltage (raw signal) which must not be confused with the polarization voltage.
Recalculate slopeSelect Recalculate slope (Recalc. slope) in the Mark information window to restore data points in the marked section of the active Flux / Slope plot, if Delete points or Interpolate points was used before. The entire plot is recalculated, such that other marked sections which may have been deleted are also restored. Compare Restore points.
Restore pointsSelect Restore points in the Mark information window to restore data points in the marked section of the active signal plot, if Delete points or Interpolate points was used before. Compare Recalculate slope.
Run DL-Protocol/Set O2 limitDL-Protocols (DLP) can be selected in DatLab 7 in the pull-down menu 'Protocol': Set DL-Protocol / O2 limit. A DL-Protocol defines the sequence of Events and Marks. Linked to DL-Protocols are templates for storing exported data in a database and for data analysis. A DL-Protocol can be assigned to O2k-Chamber A or B, or both. Instrumental DL-Protocols are used for calibrations and instrumental quality control, without experimental sample in the incubation medium. DL-Protocols for substrate-uncoupler-inhibitor titrations (see MitoPedia: SUIT) provide a guide through a sequence of coupling control states and Electron transfer-pathway states. The Titration-Injection-microPump TIP2k can be programmed for automatic control of titration steps in a DL-Protocol. A library of evaluated and tested standard DL-Protocols is provided. A Lower O2 limit [µM] can be defined for each chamber, to trigger an automatic warning when the experimental O2 concentration declines below this limit as a WARNING to remind the user that re-oxygenation of the medium may be required. Users can edit titration volumes and concentrations in the Overview window of a DL-protocol, save the overview, and export the file as a user-specific DL-Protocol [File / Export / A or B: Export DL-Protocol User (*.DLPU)].
SUIT: Browse DL-Protocols and templatesA comprehensive set of new SUIT protocols with example traces with instructions and brief explanatory texts and templates for data evaluation can be browsed from inside DatLab 7.4. Click on menu [Protocols]\SUIT: Browse DL-Protocols and templates to open a folder with all the SUIT protocols provided with the USB. DatLab protocols (DLP) for different mitochondrial preparations can be chosen to assess multiple sequences of respiratory coupling and substrate states. Protocol possess unique D## codes and comprise a fixed sequence of events and marks which cannot be changed by the user. However, the user may edit concentrations and titration volumes of injections and store the modified protocol as user-specific DL-Protocol [File]\Export\DL-Protocol User (*.DLPU). For user support, we also provide PowerPoint files with explanatory cartoons and excel templates for data evaluation. If users wish to alter the nature of the chemicals used or the sequence of injections, we ask them to contact support@oroboros.at.
SUITbrowserUse the SUITbrowser to find the best substrate-uncoupler-inhibitor-titration (SUIT) protocol for your research questions.
Open the SUITbrowser: http://suitbrowser.oroboros.at/
Each SUIT protocol receives a score from 0 to 3 in different categories that correspond to a research question (e.g. information on coupling control, information on fatty acid oxidation, among others). This score is set depending on the information which one can get from each SUIT protocol (3: this SUIT protocol is ideal, highly informative for the specific question; 2: information provided; 1: information provided, but is not a SUIT protocol mainly focused for the study of this question; 0: the specific information/question is not provided by the SUIT protocol). It is possible to choose multiple research questions at a time, to find the best protocols that address this combination of problems.
Sample - DatLabF3In the window Sample, information is entered and displayed for the sample (Sample type, Cohort, Sample code, Sample number, Subsample number and sample concentration). Entries can be edited at any time during the experiment real-time or during post-experiment analysis. All related results are recalculated instantaneously with the new parameters. Initially, the Edit experiment window displays information from the last file recorded and saved while connected to the O2k.



Save - DatLabCtrl+SSave a DLD file. When disconnected from the O2k, save any changes made under the identical file name overwriting the previous file. Such changes do not affect the raw data of the experiment, but relate to calibrations, experimental protocol, marks, events, and layout. Temporary backup files are generated by DatLab in the current user's temp directory, indicated by adding tmp.$$$ to the file name. These files are retained only if the PC has failed during data analysis. During data acquisition, the data are written continuously onto the file, hence backup files are not necessary under these conditions.
Save and DisconnectCtrl+F4Save and Disconnect: Stops data acquisition and disconnects from the O2k.
Save as - DatLabSave as a DLD file. When disconnected from the O2k, save the file under a different file name, optionally in a different directory.
Scaling - DatLabF6Scaling a graph in DatLab provides flexibility to vary the display of the plots and create Graph layouts. It allows viewing a data plot in differently scaled graphs, zooming the signal and time scales, and scrolling along the axes of the graph provide maximum information on the current experiment. This does not influence the format of stored data. Different ranges for the axes change the appearance of data dramatically. It is highly recommended to use reference layouts. »Compare: Select plots - DatLab.
Scaling factorScaling factor determines the multiplication factor that is applied to the time derivative of the signal.
Select plots - DatLabCtrl+F6In the pull-down menue [Graph], Select plots opens the Graph layout window 'Plots'. For each graph, the plots shown with the Y1 or Y2 axis can be selected, axis labels and line styles can be defined, the unit for the calibrated signal can be changed, Flux/Slope can be chosen to be displayed as Flux per volume or as normalized specific flux/flow, the background correction can be switched on or off, and the channel can be selectively displayed as the raw signal. Graph layouts can be selected and loaded or a Graph layout may be saved. »Compare: Scaling - DatLab.
SensitivitySensitivity refers to the response obtained for a given amount of analyte and is often denoted by two factors: the limit of detection and the limit of quantification.
Serial portSerial port describes the connection between O2k and Computer. With the USB-Cable 2.0\Type A-B connected, select Serial port in the Connection window. Depending on the O2k series, it is possible to connect with a Serial port or USB port.
Signal linesee O2k signal line
SmoothingVarious methods of smoothing can be applied to improve the signal-to-noise ratio. For instance, data points recorded over time [s] or over a range of wavelengths [nm] can be smoothed by averaging n data points per interval. Then the average of the n points per smoothing interval can be taken for each successively recorded data point across the time range or range of the spectrum to give a n-point moving average smoothing. This method decreases the noise of the signal, but clearly reduces the time or wavelength resolution. More advanced methods of smoothing are applied to retain a higher time resolution or wavelength resolution.
Status linesee O2k status line
Stirrer A on/offF11Toggles between stirrer on/off in the left O2k-chamber, returning to the pre set stirrer speed.
Stirrer B on/offF12Toggles between stirrer on/off in the right O2k-chamber, returning to the pre set stirrer speed.
Stirrer powerF11, F12Stirrer power is switched on and off during operation of the Oroboros O2k in DatLab by pressing [F11] (left chamber) and [F12] (right chamber), respectively. This is functional only with a stirrer bar added to each O2k chamber.
Stirrer testF9A stirrer test is performed in the Oroboros O2k for quick evaluation of the performance of the OroboPOS and for dynamic calibration. The stirrer test is initiated by pressing [F9] in DatLab. Stirring is stopped in both chambers and restarted after a selected period. The default period is 30 s, for experiments at 37 °C. At lower experimental temperature, this period should be prolonged (60 s at 25 °C). In the SOP (O2k Quality Control) for the O2 sensor test, the stirrer test is performed in the 'open' chamber in conjunction with Air calibration. In general, the stirrer test can be performed equally with an open or closed chamber. Upon automatic re-start of the stirrer (On), the increase of the oxygen signal should be rapid and monoexponential.
Synchronous time axesSynchronous time axes sets, if ticked, the time axes of all graphs at an identical range and offset, which is particularly useful while panning.
Temperature plot emptyProblem: Layout "01 Calibration Exp. Gr3-Temp" is selected, a third plot is displayed on the screen but it remains empty (no plot is shown). Newer versions of DatLab include pre-installed layouts which do not recognize some channel designations from older O2k series.
Text file - DatLabData to text file (*.csv) exports plots and events to a text file for further use in Excel and other programs. Events to text file (*.csv) exports all information in Events to a text file (*.csv). This file may be used as a protocol, including the comments in the Events.
Time resolutionTime resolution in respirometric measurements is influenced by three parameters: the response time of the POS, the data sampling interval and the number of points used for flux calculation.
USB portThe USB port describes the connection between O2k and Computer. With the USB cable connected, select USB port in the Connection window. Depending on the O2k series, it is possible to connect with a USB port or Serial port.
User code - DatLabA user code or name is entered upon starting DatLab. This window pops up automatically after opening DatLab. The current user is displayed in the O2k signal line and usernames are connected with personal graph layouts. Users can be renamed and deleted (with all linked personal layouts) using menu 'File\Manage users'.
Zero calibrationR0Zero calibration is together with air calibration one of the two steps of the OroboPOS calibration. It is performed in the closed chamber after all the oxygen has been removed by the addition of dithionite or by respiration of mitochondria or cells. Any incubation medium can be used for zero calibration with dithionite or sample. Unlike air calibration it is not necessary to perform a zero calibration each day.


Further links

» O2k-Manual: DatLab
» O2k-Demo experiments
» O2k-Catalogue: DatLab


Related topics
» Respirometry
» Fluorometry
» Spectrophotometry