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Difference between revisions of "Troubleshooting: fluorescence module"

From Bioblast
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== Atypical fluorescence signal ==
== Atypical fluorescence signal ==


::: '''Problem:''' The raw fluorescence signal is 0 V with and without fluorescence dyes.
::: '''Problem:''' The raw fluorescence signal is 0 V or noisy.  
:::'''Solution:''' How can you identify cause of the problem?
:::'''Solution:''' How can you identify cause of the problem?
:::: Please go through the following check-list:
:::: Please go through the following check-list:
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::::: a) the [[Illumination on/off|illumination]] is switched off in the ´[[O2k control]]´window.
::::: a) the [[Illumination on/off|illumination]] is switched off in the ´[[O2k control]]´window.
::::: b) the gain for Fluo-Sensor and the Fluo intensity are a number greater than 0 in the 'Amperometric,Amp' tab of the ´[[O2k control]]´ window.
::::: b) the gain for Fluo-Sensor and the Fluo intensity are a number greater than 0 in the 'Amperometric,Amp' tab of the ´[[O2k control]]´ window.
 
:::: 3. If all the above-mentioned settings and set up are correct and the detected signal with the Fluo-Sensor is still 0 perform the following test to localize the problem: Add distilled water or respiration medium into the O2k-chamber (without sample), 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.
:::: 4. If all the above-mentioned settings and set up are correct and the detected signal with the Fluo-Sensor is still 0 perform the following test to localize the problem: Add distilled water or respiration medium into the O2k-chamber (without sample), 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 (let it run for 5 min)
::::::* 1. run: Fluo-Sensor A in chamber A and Fluo-Sensor B in chamber B (let it run for 5 min)
::::::* 2. run: Fluo-Sensor A in chamber B and Fluo-Sensor B in chamber A (let it run for 5 min)
::::::* 2. run: Fluo-Sensor A in chamber B and Fluo-Sensor B in chamber A (let it run for 5 min)
:::::: If the problem is with the Fluo-Sensor, the fluorescence signal will be abnormal with the faulty Fluo-Sensor in both in chambers. In this case, the Fluo-Sensor should be shipped to us for repairs.  
:::::: If the problem is with the Fluo-Sensor, the fluorescence signal will be abnormal with the faulty Fluo-Sensor in both chambers. 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.  
:::::: If the O2k or the O2k-LED2 Fluo-Module are defective, 0 V or noisy 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 [https://www.oroboros.at/index.php/o2k-technical-support/ 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.
::::: Please contact our [https://www.oroboros.at/index.php/o2k-technical-support/ 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'''
:::: '''Example: Defective O2k or O2k-LED2 Fluo-Module'''
:::::Provided by Hazirah Hassan, University Kebangsaan Malaysia, hazy_hassan@yahoo.com
:::::Provided by Hazirah Hassan, University Kebangsaan Malaysia, hazy_hassan@yahoo.com
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::::[[File:Hassan Hazirah 2.test.png|700px]]
::::[[File:Hassan Hazirah 2.test.png|700px]]
::::[[File:Hassan Hazirah biological sample.png|700px]]
::::[[File:Hassan Hazirah biological sample.png|700px]]
:::: 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. 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.  
:::: 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. 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.  


== Negative fluorescence signal==
== Negative fluorescence signal==
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::::::* 1. run: Fluo-Sensor A in chamber A and Fluo-Sensor B in chamber B (let it run for 5 min)
::::::* 1. run: Fluo-Sensor A in chamber A and Fluo-Sensor B in chamber B (let it run for 5 min)
::::::* 2. run: Fluo-Sensor A in chamber B and Fluo-Sensor B in chamber A (let it run for 5 min)
::::::* 2. run: Fluo-Sensor A in chamber B and Fluo-Sensor B in chamber A (let it run for 5 min)
:::::: The problem is related to one of the Fluo-Sensors, if you observe a negative signal in both chambers after swapping them. The negative signal might be related to a wet plug of the Fluo-Sensor. The solution is to dry it out with ''e.g.'' a hair dryer and let it run connected to the O2k (25 °C, illumination off, distilled water in the O2k-chamber, gain for Fluo-Sensor 1000, fluorescence intensity=500) for a couple of days in a dry and warm environment. If this treatment does not solve the problem and you get still negative fluorescence values, the Fluo-Sensor should be sent back to us for further investigation and replacement. 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.  
:::::: The problem is related to one of the Fluo-Sensors, if you observe a negative signal in both chambers after swapping them. The negative signal might be related to a wet plug of the Fluo-Sensor. The solution is to dry it out with ''e.g.'' a hair dryer and let it run connected to the O2k (25 °C, illumination off, distilled water in the O2k-chamber, gain for Fluo-Sensor 1000, fluorescence intensity=500) for a couple of days in a dry and warm environment. If this treatment does not solve the problem and you get still negative fluorescence values, the Fluo-Sensor should be sent back to us for further investigation and replacement. 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.  


== Fluorescence signal is constantly at 10 V==
== Fluorescence signal is constantly at 10 V==
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::::* [[O2k-Fluo_Smart-Module#Stoppers|Black PEEK stoppers]] must be used.
::::* [[O2k-Fluo_Smart-Module#Stoppers|Black PEEK stoppers]] must be used.
::::* The fluorescence intensity cannot be set too high. Check in the [[Amperometric,Amp]] tab of the ´[[O2k control]]´ window.
::::* The fluorescence intensity cannot be set too high. Check in the [[Amperometric,Amp]] tab of the ´[[O2k control]]´ window.
::: If all the above-mentioned settings and set up are correct and the detected signal is still at 10 V (out of scale), the O2k or/and the Fluo-Sensors should be shipped to us.
::: Please contact our [https://www.oroboros.at/index.php/o2k-technical-support/ 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.
[[File:Questions.jpg|left|40px]]
[[File:Questions.jpg|left|40px]]
== Useful links ==
== Useful links ==

Revision as of 13:30, 18 March 2021


                  


O2k-Open Support

Troubleshooting: fluorescence module



Template NextGen-O2k.jpg


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



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 or noisy.
Solution: How can you identify cause of the problem?
Please go through the following check-list:
1. Check that 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).
2. In DatLab check that:
a) the illumination is switched off in the ´O2k control´window.
b) the gain for Fluo-Sensor and the Fluo intensity are a number greater than 0 in the 'Amperometric,Amp' tab of the ´O2k control´ window.
3. If all the above-mentioned settings and set up are correct and the detected signal with the Fluo-Sensor is still 0 perform the following test to localize the problem: Add distilled water or respiration medium into the O2k-chamber (without sample), 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 (let it run for 5 min)
  • 2. run: Fluo-Sensor A in chamber B and Fluo-Sensor B in chamber A (let it run for 5 min)
If the problem is with the Fluo-Sensor, the fluorescence signal will be abnormal with the faulty Fluo-Sensor in both chambers. 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 noisy 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
Hassan Hazirah 1.test.png
Hassan Hazirah 2.test.png
Hassan Hazirah biological sample.png
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. 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.

Negative fluorescence signal

Problem: The fluorescence signal of one Fluo-Sensor 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
Anne Laure Charles files.png
Solution: Add distilled water or respiration medium (without sample) 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 (let it run for 5 min)
  • 2. run: Fluo-Sensor A in chamber B and Fluo-Sensor B in chamber A (let it run for 5 min)
The problem is related to one of the Fluo-Sensors, if you observe a negative signal in both chambers after swapping them. The negative signal might be related to a wet plug of the Fluo-Sensor. The solution is to dry it out with e.g. a hair dryer and let it run connected to the O2k (25 °C, illumination off, distilled water in the O2k-chamber, gain for Fluo-Sensor 1000, fluorescence intensity=500) for a couple of days in a dry and warm environment. If this treatment does not solve the problem and you get still negative fluorescence values, the Fluo-Sensor should be sent back to us for further investigation and replacement. 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.

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. Information on how to mount the filter cap- » here.
  • Black PEEK stoppers must be used.
  • The fluorescence intensity cannot be set too high. Check in the Amperometric,Amp tab of the ´O2k control´ window.
If all the above-mentioned settings and set up are correct and the detected signal is still at 10 V (out of scale), the O2k or/and the Fluo-Sensors should be shipped to us.
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.
Questions.jpg

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