Troubleshooting: fluorescence module

                  

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Troubleshooting: fluorescence module

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

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In the following page, the most common problems occurring with either the O2k-Fluo Smart-Module or O2k-Fluo LED2-Module are summarized. The problem can be related to (a) the fluorescence settings in DatLab, (b) the fluorescence sensors, (c) the Fluorescence-Control Unit (in the case of the O2k-Fluo LED2-Module), or (d) the O2k.

Atypical fluorescence signal

Problem: The raw fluorescence signal is 0 V with and without fluorescence dyes.

Solution: How can you identify cause of the problem?

Please go through the following check-list:

1. Check that the illumination is switched off in the ´O2k control´window.

2. Check the settings for the Amperometric,Amp channel in the ´O2k control´ window, in the 'Amperometric,Amp' tab. Gain for Fluo-Sensor and the Fluo intensity cannot be 0.

3. The Fluo-Sensors are connected to the O2k and inserted into the O2k-chamber in the right position (-See: O2k-Fluo_Smart-Module#Connect_Fluorescence-Sensor_to_O2k).

4. If all the above-mentioned steps are checked and the detected signal with your Fluo-Sensor is 0 (negative or very high) perform the following test to localize the problem: Add distilled water or respiration medium into the O2k-chamber, insert the black stopper and change the settings for fluorometric measurements: Gain: 1000; Fluo intensity (Serie H or higher) or Polarisation voltage (Series D-G): 500.

• 1. run: Fluo-Sensor A in chamber A and Fluo-Sensor B in chamber B
• 2. run: Fluo-Sensor A in chamber B and Fluo-Sensor B in chamber A

If your Fluo-Sensor A is defective, the fluorescence signal should be 0 V with Fluo-Sensor A both in chamber A and B. In this case, the Fluo-Sensor should be shipped to us for repairs.

If the O2k or the O2k-LED2 Fluo-Module are defective, 0 V or negative fluorescence signal can be observed just in one chamber with two different Fluo-Sensors (see example below). In this case, the O2k or the Fluorescence-Control Unit (Series D-G) should be shipped to us for repairs.

Please contact our O2k-Technical Support to analyze your problem before shipment. Be sure to always send us original DatLab files with the above-mentioned tests to evaluate Fluo-Sensor performance.

Example: Defective O2k or O2k-LED2 Fluo-Module

Provided by Hazirah Hassan, University Kebangsaan Malaysia, hazy_hassan@yahoo.com

After changing the Fluo-Sensors between the O2k-chambers, the problem is related to the O2k or O2k-LED2 Fluo-Module since the fluorescence signal is negative and unstable in chamber B with both Fluo-Sensors. In the third figure, the fluorescence signal is very unstable in chamber B, which makes the experiment hard to interpret. Both the O2k and the LED2 Fluo-Module should be shipped to our electronic workshop.

Negative fluorescence signal

Problem: The fluorescence signal of the Fluo-Sensor in chamber B is negative, while the other Fluo-Sensor has ~0 V.

Example: Provided by Anne Laure Charles, FR_Strasbourg_Zoll J,anne laure charles <anne.laure.charles@outlook.fr

Solution: Add distilled water or respiration medium into the O2k-chamber, insert the black stopper, and change the settings for fluorometric measurements: Gain: 1000; Fluo intensity (Serie H or higher) or Polarisation voltage (Series D-G): 500.

• 1. run: Fluo-Sensor A in chamber A and Fluo-Sensor B in chamber B
• 2. run: Fluo-Sensor A in chamber B and Fluo-Sensor B in chamber A

The problem is related to Fluo-Sensor A, if you observe a negative signal in both chambers after swapping the Fluo-Sensors. The negative signal might be related to a wet plug of the Fluo-Sensor. The solution is to dry it out with e.g. hair dryer and let it run connected to the O2k for a couple of days in a dry and warm environment. If these treatments cannot solve the problem and you get still negative fluorescence values, Fluo-Sensor should be sent back to us and our electronic partner will look at it and replace it if needed. Please contact us again before sending the instrument or fluorescence sensors to us.

Fluorescence signal is constantly at 10 V

Problem: The fluorescence signal is constantly at 10 V when using the O2k-Fluo Smart-Module or O2k-Fluo LED2-Module.

Solution: This is probably due to high light exposure to the photodiode. Check the following parameters:

• The chamber illumination must be switched off: Check it in the O2k control window.
• The photodiode filters must be inserted, otherwise the light emitted from the LED will not be filtered. Click here for more information on how to mount the filter cap.
• Ensure that the Smart Fluo-Sensor is correctly inserted at the O2k frame of the chamber window.
• Use black PEEK stoppers.
• Check the fluorescence intensity in the ´O2k control´ window, in the Amperometric,Amp tab. It might be possible that the fluorescence intensity is too high.

Useful links

» Amplex UltraRed

» Safranin

» TMRM

» Rhodamine 123

» Magnesium Green

» Calcium Green

» O2k-FluoRespirometer manual

» O2k-Fluo LED2-Module

» O2k-Demo experiments

» Sole source information

» Oroboros O2k