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O2k-Fluo LED2-Module

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O2k-Fluo LED2-Module

O2k-Catalogue

Description The O2k-Fluorescence LED2-Module is an amperometric add-on module to the O2k-Core, adding a new dimension to HRR. Optical sensors are inserted through the front window of the O2k-glass chambers, for measurement of hydrogen peroxide production (Amplex red), ATP production (Magnesium green), mt-membrane potential (Safranin), Ca2+ (Calcium green), and numerous other applications open for O2k-user innovation.

The O2k-Fluorescence LED2-Module consists of optical sensors for both O2k-Chambers (LEDs for green and blue excitation), optical filters, Fluorescence-Control Unit for regulation of light intensity, data input into the O2k-Main Unit, and the updated DatLab software.

Product ID 12100-01
Type O2k, O2k-Module, MultiSensor, Catalogue
Link O2k-Fluorescence@OROBOROS, Oxygraph-2k, G MiPNet17.05 O2k-Fluorescence LED2-Module
Image
O2k-Fluorometer Series G.jpg

The O2k-Fluorescence LED2-Module consists of

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O2k-Fluorometry Workshops


O2k-Manual: O2k-Fluorescence LED2-Module

>> O2k-Manual: O2k-Fluorescence LED2-Module
Open Innovation: The Manual for the O2k-Fluorescence LED2-Module evolves as a guided tour through the O2k-Catalogue: O2k-Fluorescence LED2-Module.

Scope of Application

There are many different fluorometric measurement techniques. The O2k-Fluorescence LED2-Module is designed for measurements that are typically performed by placing a (stirred) cuvette with the sample into a (spectro)fluormeter. Protocols for these cuvette based measurements can be implemented with the O2k-Fluorescence LED2-Module, adding simultaneous measurement of respiration and the monitoring of oxygen concentration to the protocol. The scope of possible applications is further defined by the excitation wavelengths provided by the sensors Fluorescence-Sensor Green and Fluorescence-Sensor Blue and the availability of suitable emission filters. Therefore, classical fluorometric measurements can be “translated” to an O2k equipped with the O2k-Fluorescence LED2-Module if the following conditions are met:

  • the fluorescence measurement was done in a cuvette using a (spectro)fluorometer
  • one of the fluorophores listed below under O2k-Fluorescence_LED2-Module#Application-specific_settings was used OR excitation and emission wavelength of the fluorophore are suitable for use with the O2k-Fluorescence LED2-Module. Please contact Oroboros Instruments if in doubt about the latter point.

The O2k-Fluorescence LED2-Module is not designed to fulfill the functions of other devises used in fluorometric measurements e.g. fluorescence microscopes, flow cytometers, etc..

Setup of the O2k-Fluorescence LED2-Module

  1. Setup of the O2k-Fluorescence LED2-Module
  2. Selecting a Fluorescence Sensor
  3. Mounting a Filter-Cap
  4. Connect Fluorescence-Sensor to O2k-Main unit

Electronic settings

  1. Power on
  2. LED-intensity
  3. Amplification or Gain

Application-specific settings

The light intensity of the LED (LED-intensity) and the signal amplification (Gain) can be adjusted in a wide range. The table suggests initial values, which may be optimised for specific applications.

Application Sensor Filter set Light intensity (polarization voltage) - Note a Gain
Amplex® UltraRed Fluorescence-Sensor Green AmR 100 - 500 1000

(at light intensity = 100)

TMRM Fluorescence-Sensor Green AmR 500 - 1000 1000
Safranin Fluorescence-Sensor Blue Saf 200 for [safranin] > 2 µM; 500 or higher for [safranin] < 2 µM 1000
Magnesium green Fluorescence-Sensor Blue MgG / CaG 300 Note b
Calcium green Fluorescence-Sensor Blue MgG / CaG Note b
  • Note a: Set the polarization voltage [mV] for the amperometric channel (Amp) in the DatLab menu [O2k-MultiSensor \ O2k Control \ Amp polarisation voltage]. Divide the polarisation voltage [mV] by 100 to obtain the current [mA] through the LED. For simple operation instructions, it is sufficient to refer to the polarization settings selected in DatLab.
  • Note b: The amplification for Magnesium green(R) and Calcium green(R) depends on the concentration of the fluorophore, which varies drastically between different applications. Therefore, no general recommendation is given for the appropriate gain.


DatLab-Analysis

Additional Templates for Fluorescence

A DatLab Template file for fluorescence applications can be downloaded @OROBOROS. See MiPNet12.07 for instructions how to import a DatLab Template files.


Observing the fluorescence signal

Use Graph Layout “A Amp” to display “Amp Raw Signal”

Check Amp raw signal form Graph/Select Plots to display the fluorometric signal
Graph / Select plots‎

Graph layout: Three plots are available in DatLab based on the recorded signal: Amp Raw Signal, Amp Calibrated, and Amp Slope. These plots can be selected from the drop-down lines and displayed with their check boxes either on the Y1 or Y2 [Graph layout / Select Plots].


Amp Raw Signal displays the raw voltage (including amplification) as recorded by the Oxygraph at a given gain setting.

Amp Calibrated is the signal after calibration with the parameters set in the O2k-MultiSensor Calibration window.

Amp slope is the time derivative of the calibrated signal, multiplied by 1000, in units [m(conc. Unit during calibration)/s], so if the signal was calibrated in µM [nmol/ml] the unit of the slope is pmol/(s ml). To obtain the slope of the raw signal check the appropriate box in the calibration window (DatLab 5.1.0.130 and above).

Graphs can be generated to display oxygen and fluorescence data, or several graphs can be added to display oxygen and fluorescence data separately. Layout templates are provided, which can be modified and saved as appropriate. All graph settings can be saved as user-defined layouts MiPNet12.07.

The calibration window

If the active plot is "Amp" or "Amp slope" using either [MultiSensor]/[MultiSensor Calibration], or [Ctrl+F5] , pressing the right mouse key on the plot legend will open the calibration window for the Amp channel, see MiPNet12.08.

Setting properties of calibrated signal plot and slope plot

DL5 1 0 130 Amp calibration user settings.PNG
  • Type the desired plot name in the field "Name for Amp-channel". Avoid long names.
  • Choose the desired unit for the calibrated signal from the drop down menu beside "Unit". Default: "µM".
  • Choose the desired factor for slope calculation from the drop down menu beside "Slope factor". Default: 1000. The correct unit for the slope will be set by DatLab depending on the chosen unit for the calibrated signal and the factor for slope calculation. Changing the factor will recalculate the values for the slope plot correctly.
  • To calculate the slope based on the raw signal, activate the checkbox "Calculate from raw signal". If viewing the raw (uncalibrated signal) it is recommended to use this option.
  • Click "Calibrate and Copy to Clipboard" to apply all changes.

Two point linear calibration: To perform a simple two-point linear calibration of the "Amp channel"

  • select "Amp" as active plot
  • place two marks on to stable regions of the signal corresponding to two known concentrations
  • open the calibration window
  • use the drop down menus in the "Select Mark" column to to select the two marks of known concentration
  • enter the concentrations in the column labeled "Amp concentration"; express the concentrations in the unit selected as described above
  • Click "Calibrate and Copy to Clipboard" to apply all changes.


Multiple point linear calibration:

  1. Calculate the regression of raw voltage [V] as a function of concentration [µM] in a spreadsheet program.
  2. Note slope and intercept; add the two values to get the value for "slope+intercept"
  3. Open the MultiSensor calibration window
  4. Enter the data matrix shown below
  5. Press Calibrate and Copy to clipboard.
DL5 Amp multpoint calib.PNG
c [µM] Select Mark Raw Signal [V]
1 leave empty slope + intercept
0 leave empty intercept



Alternative: If the regression was done for c in [µM] against raw voltage in [V] the following data matrix has to be entered:

c [µM] Select Mark Raw Signal [V]
slope + intercept leave empty 1
intercept leave empty 0

Performing an experiment

The O2k-Fluorescence Sensors can be inserted into the chamber of the O2k at any time when using the O2k.

  1. Set up the instrument as described O2k-Fluorescence_LED2-Module#Setup_of_the_O2k-Fluorescence_LED2-Module above.
  2. Switch off the chamber light [F10].
  3. Observe the "Amp raw signal" and "slope Amp" as described above. It will take some minutes for the sensor to reach a constant temperature and therefore a stable signal. Therefore, it is advisable to insert the fluorescence sensors early in the set up of the instrument. However, they can be removed at any time to visually check the chamber. When a sensor is removed for a short time only it will reach a stable temperature and therefore a stable signal very soon after re-inserting it into the window of the O2k chamber.
  4. Set up your experiment as usually for respiration experiments. Remove the fluorescence sensors whenever necessary. The time for gaining a stable oxygen signal at open chamber can be used to thermally equilibrate the fluorescence sensors.
  5. After the chambers are closed and a visual check showed no bubbles (remove the fluorescence sensors from the chamber window for this), switch off the O2k chamber light and start the experiment. You will probable want to observe both the fluorescence channels (Amp) and the oxygen channels. Some initial graph layouts are provided in the additional templates for fluorescence.

Calibration

Different fluorescence applications require very different calibration procedures. For some types of calibrations it will be the best approach to set marks on the "Amp raw signal" and export these to a spreadsheet. Note that for some applications (H2O2 production) the slope of the fluorescence signal, not the signal itself, will be the parameter of biological importance.


Linear Calibration

For the application Amplex(R) (Ultra)red a calibration template is available with detailed instructions: O2k-Fluorescence. The following text provides more general guidelines.

If there is a linear relationship between fluorescence emission and concentration typically a multiple-point calibration is performed, plotting the signal as a function of concentration over a wide concentration range. The obtained regression parameters (slope and intercept) may be used either in a spreadsheet program to calculate averaged concentrations or used via the DatLab calibration window to directly display concentrations. Two point calibrations can be done directly in DatLab, see DatLab-Analysis for both options. For some applications calibrations may be easily done using the Titration-Injection-microPump (TIP2k).


Data export and linear calibration: Mark stable sections on the raw signal, use or generate a template of mark names, and copy to clipboard in Marks Statistics [F2]. Copy into an Excel template for linear regression. This template can be modified according to the specific calibration experiment (titration volumes, concentrations, number of data points, …). Perform a linear regression of the raw signal as a function of analyte concentration. For highest accuracy, only the concentration range used in the final experiment should be included in the regression. Obtain the regression parameters (slope and intercept).

First time users

  • Set up the instrument as described above.
  • Get familiar with the instrument and the software options:
    • With Fluorescence-Sensors outside the chamber, use DatLab to vary the light intensities of the light source in the sensor. For the blue and green sensor make sure that you see the change in light intensity with your own eyes, but without directly looking into the light source.
    • Insert the Fluorescence-Senors into the chamber and make sure that you can observe the change in the Amp signal when changing the light intensity. If the signal is constant near to 10 Volts the reasons may be a too high light intensity, a too high gain or a (yellow) chamber light that is still switched on.
    • Play with light intensity and Amp gain and observe the changing signal in DatLab
    • Vary the scaling of the Amp plot
  • Perform a calibration experiment without biological sample for your application. Make sure you observe the expected change in signal
  • Run a blank experiment to access potential non-biologic effects of the substances you plan to use in your experiment on the observed fluorescence signal.

References

See also


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MitoPedia methods: Fluorometry