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Difference between revisions of "MiPNet17.05 O2k-Fluo LED2-Module"

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* Contribution to K-Regio project ''MitoCom Tyrol'', funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).       >> [[MitoCom|''MitoCom O2k-Fluorometer'']]
* Contribution to K-Regio project ''MitoCom Tyrol'', funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).       >> [[MitoCom_O2k-Fluorometer|''MitoCom O2k-Fluorometer'']]
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== O2k-Manual: O2k-Fluo LED2-Module ==
== O2k-Manual: O2k-Fluo LED2-Module ==

Revision as of 08:34, 15 June 2015

                



MiPNet17.05 O2k-Fluo LED2-Module

Publications in the MiPMap
O2k-Manual
O2k-Fluo LED2-Module. »Bioblast pdf«

» Versions

OROBOROS (2015-06-09) Mitochondr Physiol Network

Abstract: Fasching M, Gradl P, Gnaiger E (2015) O2k-Fluo LED2-Module. Mitochondr Physiol Network 17.05(08):1-6.


O2k-Manual: The O2k-Fluo LED2-Module is a modular extension of the O2k-Core (Series D upwards). A growing number of fluorescence markers enables determination of diverse mitochondrial processes in addition to oxygen consumption, including generation of H2O2, ATP production, mitochondrial membrane potential and Ca2+, extendable by user-specific applications.

» Product: O2k-Fluorometer, O2k-Fluo LED2-Module, O2k-Catalogue

Keywords: HRR, Fluorometry

O2k-Network Lab: AT_Innsbruck_OROBOROS


Labels: MiParea: Respiration, Instruments;methods 





HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Manual, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 

O2k-MultiSensor 


  • Contribution to K-Regio project MitoCom Tyrol, funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).    >> MitoCom O2k-Fluorometer

O2k-Manual: O2k-Fluo LED2-Module


Template NextGen-O2k.jpg


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



Setup of the O2k-Fluo LED2-Module

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


LED-intensity and amplification

  1. LED-intensity
  2. Amplification

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 can be optimised for specific applications.

  • The settings depend on the concentration of the fluorophore, which vary between different applications. Therefore, only recommendations for specific fluorophore concentrations are given. In the Amplex Ultrared assay the fluorophore is formed during the experiment.
  • The recommendations apply to experiments at 37 °C. The Fluorescence intensity increases strongly at lower temperatures. Then the light intensity is reduced to avoid off-scale signals.


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

(at light intensity = 100)

TMRM Fluorescence-Sensor Green AmR 200 - 500 1000 at c(TMRM) = 2 µM
Safranin Fluorescence-Sensor Blue Saf 100 - 200 for c(safranin)= 2 µM; 1000 at c(Mg Green) = 2 µM,
Magnesium green Fluorescence-Sensor Blue MgG / CaG 100 - 300 1000 at c(Mg Green) = 2 µM
Calcium green Fluorescence-Sensor Blue MgG / CaG 100 -300 1000 at c(Ca Green) = 2 µM
  • 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.


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 O2k 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 a unit for the calibrated signal from the drop down menu beside "Unit". Default: "µM".
  • Choose the 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


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).


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-Fluo_LED2-Module#Setup_of_the_O2k-Fluo_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.


Fluorescence and the TIP2k

Fluorescence methods, especially when requiring multiple point calibrations, open up some new applications for the Titration-Injection microPump (TIP-2k). However, there is one caveat: The needle of the TIP may itself influence the optical signal. There are several ways how this can be dealt with:

  • A test shows that the signal is not affected by the presence/ absence of the TIP needle. I presume such a check will show that there is no problem anyway.
  • The TIP needles stay in the chambers for the rest of the experiment and are only temporary removed for other injections.
  • The opposite approach: The needle is removed between the calibration injections to record sequences of signals without the influences of the TIP. Marks from these sections are used for the calibration procedure. After the calibration the TIP needle can be removed permanently from the chamber.
  • Not related to calibrations: In applications in which the absolute signal is not so important (like Amplex, where only the slope matters) there should be no problem to use the TIP for some parts of the experiment (unless even the sensitivity is changed by the needle in a significant way, which seems improbable).