Talk:O2k-Fluo LED2-Module

Oxygraph Series A to C

Question: Why can only one chamber be used for fluoresecence measurements with O2k Series A to C?

Answer: The oxygen channel from one chamber will be used to record the fluorescence signal form the other channel. Therefore only signals from one chamber can be recorded.

Question: What are the performance limitations when using the O2k-Fluorescence Module with O2k Serial A to C?

Answer:

• Only one chamber can be used for simultaneous fluorescence and respiration measurements.
• The amplification of the fluorescence signal is done outside the oxygraph in a special amplification box. The resulting signal stability is usually good and the same approach has been used to measure NO for O2k series A to C. However, signal stability may not reach the high level achieved when using O2k Series D and above.
• Performing the measurement and analyzing the data is a little bit different from the way it can be done with O2k Series D and above. The fluorescence Signal will actually be found in the signal labeled "oxygen" from the unused chamber and different amplification levels will be available. The situation is very similar to measuring NO with O2k Series A to C, see H MiPNet15.05 NO-Manual.

Fasching Mario 11:55, 25 January 2012 (CET)

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

--Fasching Mario 10:49, 23 February 2012 (CET)

Coupling of the O2k to optical instruments: Geometry

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 However, in Tony Hickey's (Hickey 2011 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 Duran optical properties The “via the window” approach has several important benefits:

1. the optical sensor does not “see” 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.
2. no physical contact of the sample with the optical sensor necessary
3. 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

The black but otherwise regular stopper is required to shield the chamber from outside light. 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. We used this configuration not only with our LED based fluorescence module but also to couple a full spectrofluorometer to the O2k chamber. 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. --Fasching Mario 09:26, 17 February 2012 (CET)