Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

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

From Bioblast
(44 intermediate revisions by 11 users not shown)
Line 1: Line 1:
{{OROBOROS navigation line page name}}
{{Template:OROBOROS support page name}}
{{Publication
{{Publication
|title=[[Image:O2k-Manual.jpg|right|70px|link=O2k-Manual|O2k-Manual]] O2k-Fluo LED2-Module. [[Media:MiPNet17.05 O2k-Fluoresence LED2-Module.pdf|»Bioblast pdf«]]
|title=[[Image:O2k-Manual.jpg|right|70px|link=O2k-Manual|O2k-Manual]] O2k-Fluo LED2-Module.
|info=[http://www.bioblast.at/index.php/File:MiPNet17.05_O2k-Fluoresence_LED2-Module.pdf Versions]
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/9/9e/MiPNet17.05_O2k-Fluo_LED2-Module.pdf |Bioblast pdf]] »[http://wiki.oroboros.at/index.php/File:MiPNet17.05_O2k-Fluo_LED2-Module.pdf Versions]
|authors=OROBOROS
|authors=Oroboros
|year=2015-05-21
|year=2016-08-09
|journal=Mitochondr Physiol Network
|journal=Mitochondr Physiol Network
|abstract=Fasching M, Gradl P, Gnaiger E (2015) O2k-Fluo LED2-Module. Mitochondr Physiol Network 17.05(07):1-7.
|abstract=Fasching M, Gradl P, Gnaiger E (2016) O2k-Fluo LED2-Module. Mitochondr Physiol Network 17.05(09):1-6.  
 
{{MiPNet pdf page linking to MitoPedia}}
 
:» Product: [[O2k-Fluo LED2-Module]], [[Oroboros O2k-Catalogue |O2k-Catalogue]]
'''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 H<sub>2</sub>O<sub>2</sub>, ATP production, mitochondrial membrane potential and Ca<sup>2+</sup>, extendable by user-specific applications.
|mipnetlab=AT_Innsbruck_Oroboros
:» Product: [[O2k-Fluorometer]], [[O2k-Fluo LED2-Module]], [[O2k-Catalogue OROBOROS |O2k-Catalogue]]
|keywords=[[HRR]], [[Fluorometry]]
|mipnetlab=AT_Innsbruck_OROBOROS
}}
}}
{{Labeling
{{Labeling
|area=Respiration, Instruments;methods
|area=Respiration, Instruments;methods
|instruments=Oxygraph-2k, O2k-Fluorometer, O2k-Manual, Protocol
|instruments=Oxygraph-2k, O2k-Fluorometer, O2k-Manual, O2k-Protocol
|additional=O2k-MultiSensor
|additional=O2k-MultiSensor, DatLab, Archive
}}
}}
* Contribution to K-Regio project ''MitoCom Tyrol'', funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).&nbsp;&nbsp;&nbsp;    >> [[MitoCom_O2k-Fluorometer |''MitoCom O2k-Fluorometer'']]
__TOC__
== O2k-Manual: O2k-Fluo LED2-Module ==
{{Technical support integrated}}
{{#set:Technical service=NO (amperometric) signal |Technical service=O2k-Fluorometer}}
=== Setup of the O2k-Fluo LED2-Module ===
# [[Fluorescence-Control_Unit#Setup_of_the_O2k-Fluorescence_LED2-Module|Setup of the O2k-Fluo LED2-Module]]
# [[Fluorescence-Sensor#Select_the_Fluorescence-Sensors|Selecting a Fluorescence Sensor]]'''
# [[Filter-Cap#Mounting_a_Filter-Cap|Mounting a Filter-Cap]]
# [[Fluorescence-Sensor#Connect_Fluorescence-Sensor_to_O2k|Connect Fluorescence-Sensor to O2k-Main unit]]
# [[Fluorescence-Control_Unit#Power_on|Power on]]
=== LED-intensity and amplification ===
# [[Fluorescence-Control_Unit#Control_of_LED-intensity|LED-intensity]]
# [[Fluorescence-Control_Unit#Gain|Amplification]]
The light intensity of the LED ([[Fluorescence-Control_Unit#Control_of_LED-intensity|LED-intensity]]) and the signal amplification ([[Fluorescence-Control_Unit#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.
{| class="wikitable"
|-
! Application !!Sensor!! Filter set !! Light intensity (polarization voltage) - ''Note a''  !! Gain !! Comment
|-
| [[Amplex® UltraRed]]||[[Fluorescence-Sensor Green]] || [[ Filter Set AmR| AmR]] || 100 - 500 || 1000 <br />
(at light intensity = 100)
|-
| [[TMRM]]||[[Fluorescence-Sensor Green]] || [[ Filter Set AmR| AmR]] ||  200 - 500|| 1000 || at c(TMRM) = 2 µM
|-
| [[Safranin]] ||[[Fluorescence-Sensor Blue]]|| [[Filter Set Saf|Saf]] || 100 - 200 for c(safranin)= 2 µM; || 1000 || at c(Mg Green) = 2 µM,
|-
| [[Magnesium green]]||[[Fluorescence-Sensor Blue]]|| [[Filter Set MgG / CaG| MgG / CaG]] || 100 - 300|| 1000 || at c(Mg Green) = 2 µM
|-
| [[Calcium green]]||[[Fluorescence-Sensor Blue]]|| [[Filter Set MgG / CaG| 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 [http://www.oroboros.at/?O2k-fluorescence @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”
[[Image:DL5_graph_select_plots_amp.PNG|thumb|250px|alt=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'''<nowiki>. 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].</nowiki>
'''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  <nowiki>[Ctrl+F5] </nowiki>, 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'''
[[Image:DL5_1_0_130_Amp_calibration_user_settings.PNG‎|400px|right]]
* 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:'''
# <nowiki>Calculate the regression of raw voltage [V] as a function of concentration [µM] in a spreadsheet program.</nowiki>
# Note slope and intercept; add the two values to get the value for "slope+intercept"
# Open the MultiSensor calibration window
# Enter the data matrix shown below
# Press Calibrate and Copy to clipboard.
[[file:DL5_Amp_multpoint_calib.PNG|300px|right]]
{| class="wikitable"
|-
! c [µM] !! Select Mark !! Raw Signal [V]
|-
| 1 || leave empty || slope + intercept
|-
| 0 || leave empty|| intercept
|}
<nowiki>Alternative: If the regression was done for c in [µM] against raw voltage in [V] the following data matrix has to be entered:</nowiki>
{| class="wikitable"
|-
! 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 (H<sub>2</sub>O<sub>2</sub> 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: [http://www.oroboros.at/?O2k-Fluorescence 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 [[O2k-Fluorescence LED2-Module#DatLab-Analysis| DatLab-Analysis]] for both options.
For some applications calibrations may be easily done using the Titration-Injection-microPump (TIP2k).
'''Data export and linear calibration:''' <nowiki>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 </nowiki>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.
# Set up the instrument as described [[O2k-Fluo_LED2-Module#Setup_of_the_O2k-Fluo_LED2-Module above.]]
# Switch off the chamber light [F10].
# 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.
# 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.
# 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).
=== First time users ===
* Set up the instrument as described [[O2k-Fluo_LED2-Module#Setup_of_the_O2k-Fluo_LED2-Module| above.]]
* Get familiar with the instrument and the software options:
**  With [[Fluorescence-Sensor]]s 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.

Revision as of 21:59, 13 February 2020


                  


O2k-Open Support

MiPNet17.05 O2k-Fluo LED2-Module


Publications in the MiPMap
O2k-Manual
O2k-Fluo LED2-Module.

» Bioblast pdf »Versions

Oroboros (2016-08-09) Mitochondr Physiol Network

Abstract: Fasching M, Gradl P, Gnaiger E (2016) O2k-Fluo LED2-Module. Mitochondr Physiol Network 17.05(09):1-6.

O2k-technical support and open innovation
Open the pdf document above.
» Current O2k-series: NextGen-O2k Series XB and O2k Series J
» Current software versions DatLab 8.0: MitoPedia: DatLab
» Product: O2k-Fluo LED2-Module, O2k-Catalogue


O2k-Network Lab: AT_Innsbruck_Oroboros


Labels: MiParea: Respiration, Instruments;methods 





HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Manual, O2k-Protocol 

O2k-MultiSensor, DatLab, Archive