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| == Measuring hydrogen peroxide == | | == Measuring hydrogen peroxide == |
| See Measuring H2O2 production with any brand similar to Amplex(R) REed see [[Amplex red]] and especially the accompanying Discussion page. | | See Measuring H2O2 production with any brand similar to Amplex(R) REed see [[Amplex red]] and especially the accompanying Discussion page. |
| For an overview of methods to mneasure H2O2 concentration or H2O2 production see [[Measuring hydrogen peroxide]]. | | For an overview of methods to mneasure H2O2 concentration or H2O2 production see [[Measuring hydrogen peroxide]]. |
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| The O2k-system is very flexible. If you take cultured cells or isolated mitochondria, split them into the two chambers of the O2k, then you can maximize the number of simultaneously measured parameters: | | The O2k-system is very flexible. If you take cultured cells or isolated mitochondria, split them into the two chambers of the O2k, then you can maximize the number of simultaneously measured parameters: |
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| == Oxygraph Series A to C ==
| | In both chambers oxygen concentration and oxygen flux (O2k-Core; http://www.bioblast.at/index.php/O2k-Catalogue:_O2k-Core ); |
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| *The Oxygraph-2k, [[O2k-Main Unit#O2k-Series|Series]] A to C, can currently not be equipped with an O2k-Fluorescence LED2-Module. We thank you for your understanding.
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| [[User:Fasching Mario|Fasching Mario]] 11:55, 25 January 2012 (CET)
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| == Other fluorophores ==
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| '''Question:''' In the brochure, specific mention is made of Amplex Red, Mg green, Safranin, and Ca green. Will other fluorophores work (e.g., MitoTracker or MitoSOX)? Is the module tuneable at all?
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| '''Answer:''' For some application no (or little) "tuning" might be necessary, see below for your examples.
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| a.) General
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| The O2k-Fluorescence LED2-Module is "tunable" with two different levels of involvement:
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| The detected emission wavelength can be changed totally by changing the filter in front of the photo diode. Similarly, some limited fine tuning of the effective excitation wavelength range is possible by changing the filters in front of the LED. Since filters (plastic films) are easily exchangeable this procedure is open to user innovation, maybe with a little help from us.
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| In contrast, to drastically change the excitation wavelength a different sensor with a different LEd has to be used. At the moment we offer [[Fluorescence-Sensor Green]] and [[Fluorescence-Sensor Blue]]. There is limited opportunity for the customer to modify this part for herself, though we might offer a third sensors for UV excitation in the future.
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| b.) Examples:
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| Remarks: a.) light intensity always has to be optimized to make sure the fluorophore considered is not affected significantly by photo bleaching. b:) Many fluorophores are typically used for imaging applications. Be aware that the O2k-Fluorescence LED2-Module is NOT an imaging tool but quantifies fluorescence derived from the entire chamber.
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| '''MitoSOX''': According to my tables, MitoSOX has an excitation maximum of 510 nm and an emission maximum of 772 nm. I do not have excitation or emission spectra right now to check if anything unusual or weird is going on with MitoSOX but just from the wavelengths this sounds easy. You could start with [[Fluorescence-Sensor Blue]] plus both the LED filter and photo diode filters from [[Filter Set Saf]]. Due to the huge separation of excitation and emission it is probably possible to use no LED filter at all. It should be checked if this affects the form of any calibration curve in any way.
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| '''Mitotracker''': For me it would be interesting to learn the intended non-imaging application for Mitotracker? Also there are at least three different Mitotracker fluorophores: "green", orange", and "red". Which one do you want to use?--[[User:Fasching Mario|Fasching Mario]] 09:57, 21 May 2012 (CEST)
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| == Coupling the O2k to optical instruments: Geometry ==
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| For various analytical techniques it is desired to couple optical sensors and light sources to the O2k chamber. The initial approach to do so was to insert the optical probe via a custom designed black stopper from above directly into the chamber, see ⊠This approach (light and detection from above) requires also the use of black stirrer
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| However, in Tony Hickey's ([[Hickey 2012 J Comp Physiol B]]) and in our own development of the fluorescence module ([[Fasching 2011 Abstract Berlin]] it became clear that for most applications in the visible range it is a better and easier strategy to introduce the light via the front window and detect the light via the same way. The Duran glass of the O2k chamber has very high transmission down to at least 350 nm and is probable usable even further down, see [http://www.duran-group.com/en/about-duran/duran-properties/optical-properties-of-duran.html Duran optical properties ]
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| The âvia the windowâ approach has several important benefits:
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| # the optical sensor does not âseeâ the stirrers. Therefore, for comparable small optical sensor diameters there was no need to use black stirrers. In fact using white stirrers increased overall sensitivity without introducing any disturbances.
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| # no physical contact of the sample with the optical sensor necessary
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| # the space on top of the stopper is not taken up by the optical sensor, allowing for easier titrations and at least potentially for simultaneous use of additional electrodes introduced via the stopper
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| The black but otherwise regular stopper is required to shield the chamber from outside light.
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| Development with this approach was facilitated by the (rather lucky) fact that the diameter of the O2k chamber window is identical to the chamber diameter, therefore a stopper with o-ring designed to be introduced into the chamber from above, can also be placed in the chamber window for initial experiments. As a permanent, final sensor a more stable solution should be used however.
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| We used this configuration not only with our LED based fluorescence module but also to couple a full spectrofluorometer to the O2k chamber.
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| We think this might be worth to try also for these groups that need a full spectrofluorometer or other light guide based detectors / sources attached to the oxygraph.
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| --[[User:Fasching Mario|Fasching Mario]] 09:26, 17 February 2012 (CET)
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Measuring hydrogen peroxide
See Measuring H2O2 production with any brand similar to Amplex(R) REed see Amplex red and especially the accompanying Discussion page.
For an overview of methods to mneasure H2O2 concentration or H2O2 production see Measuring hydrogen peroxide.
Simultaneous measurement of TPP and fluorescence
Question: Will it be possible to measure JO2, fluorescence AND TPP simultaneously, or just JO2/fluorescence vs JO2/TPP?
Answer: Background
The main unknown is the chemical compatibility of the methods. We still have to find out whats the effect of Amplex red and its reaction product on the TPP signal.
If this is not a big problem there are technical issues: a black TPP stopper will be required and then still light entering the chamber through the TPP electrode might be a problem. Producing the TPP electrodes in black is presumable not a very good idea because then it will no longer be possible to "see" the position of the membrane. I think the technical problems are solvable but first we have to check for chemical compatibility.
Update:
First exploratory experiment:
A chamber was equipped with TPP electrodes (black stoppers) and a fluorescence module for measuring H2O2 via the Amplex Red method. A standard TPP calibration was carried out.
Amplex red / HRP addition resulted only in a minor signal in the TPP channel, a further TPP calibration showed no obvious large negative effects on the performance of the TPP electrode. An H2O2 calibration of the fluorescence signal was carried out: The sensitivity for H2O2 was reduced by a factor of 3 to 4 as compared to measurements without TPP electrodes. At the same time the noise was drastically increased. In the comparative experiment (without TPP electrodes) no random noise was seen - digital resolution was the limiting factor. Based on this comparison, noise increased in the experiment with TPP electrodes at least by a factor of 20, probable more.
It will be possible to estimate this factor with a higher precision after running an experiment without TPP electrodes at a higher gain (higher digital resolution).
It was further tested whether the presence of TPP+ alone (without electrodes) could explain this behavior but, if there were any effects at all, they were quite small.
Peroxide additions corresponding to a concentration change of 110 nM (110 pmol/ml) were still well visible but not additions corresponding to a 22 nM (22 pmol/ml) concentration change.
This was only one exploratory experiment (2 chambers)!
Based on this preliminary data:
An important point is why one would want to measure TPP and H2O2 simultaneously in the same chamber: If this is "only" for time / sample economy the results above strongly discourage this idea. Parallel measurement e.g one chamber TPP / one chamber H2O2 are preferable.
If there is a very strong need to measure both parameters on the same sample (e.g. if strong in-homogeneity among samples in this respect is suspected) and a comparable high H2O2 flux is expected the simultaneous measurement might be principally doable, especially if further optimizations are possible. E.g.: Maybe with the TPP electrode in the chamber black stirrers become important again? However, this certainly would involve some methodological work and the results would be second class at the best.
What would be your driving interest for simultaneous measurements?
best greetings
Mario
Question: How many parameters can be measured at once?
Answer:
The O2k-system is very flexible. If you take cultured cells or isolated mitochondria, split them into the two chambers of the O2k, then you can maximize the number of simultaneously measured parameters:
In both chambers oxygen concentration and oxygen flux (O2k-Core; http://www.bioblast.at/index.php/O2k-Catalogue:_O2k-Core );
