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howto:how_to_measure_the_instrument_response_function_irf [2019/11/04 09:45] – [Appropriate Count Rate for Measuring an IRF] buschmannhowto:how_to_measure_the_instrument_response_function_irf [2023/09/07 22:55] (current) – [Using samples with ultrafast decay] peter
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 ====== How to Measure the Instrument Response Function (IRF) ====== ====== How to Measure the Instrument Response Function (IRF) ======
-(For an explanation of the term IRF see [[glossary:IRF]])+(For an explanation of the term Instrument Response Function see [[glossary:IRF]]) 
 + 
 +The following video shows how to measure na IRF on confocal microscopes.
  
 {{youtube>cjSgCBe3ReM?large}} {{youtube>cjSgCBe3ReM?large}}
 +
 +\\
  
  
-==== Make sure that the detection count rate is much lower than the count rate used for fluorescence decay measurement. ====+==== Low count rate during IRF measurements is important! ====
  
-Diluting the scattering solution is better than using grey filters. Ideal is when the decay and the IRF are recorded at the same [[glossary:differential count rate]] (and NOT at the same average count rates).+Make sure that the detection count rate is much lower than the count rate during a fluorescence decay measurement. Diluting the scattering solution is better than using grey (ND) filters. Ideal is when the decay and the IRF are recorded at the same [[glossary:differential count rate]] (and NOT at the same average count rate).
  
-If the IRF should be measured on a microscope system with SPAD detectors, in the UV range also the Raman-scattering of water can be used. E.g. the Raman scattering can be recorded with a HQ480/40 bandpass filter, if a 405nm diode is used. This method is less suited for long wavelengths, as the Raman efficiency decreases. The water must be very pure in order not to capture any fluorescence.+If the IRF should be measured in the UV range on a microscope system with SPAD detectors, the Raman scattering of water can be used, too. E.g. the Raman scattering can be recorded with a HQ480/40 bandpass filter, if a 405nm laser diode is used. This method is less suited for long wavelengths, as the Raman scattering decreases. In order to avoid signal contamination by any fluorescence, the water must be very pure.
  
    
 ===== Using samples with ultrafast decay ===== ===== Using samples with ultrafast decay =====
  
-Some detectors (particularly SPADs) have wavelength dependent timing response. In this case an IRF recorded at the excitation wavelength may not be useful for precise reconvolution. The solution is to acquire the IRF at the fluorescence wavelength, or at least spectrally closer to the fluorescence emission. +Some detectors (particularly MPD SPADs) have a wavelength dependent timing response. In this case an IRF recorded at the excitation wavelength may not be useful for precise reconvolution. The solution is to acquire the IRF at the fluorescence wavelength, or at least spectrally closer to the fluorescence emission wavelength. 
  
 ==== General recipe ==== ==== General recipe ====
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  prepare 1mL of saturated water solution of KI (potassium iodide)   prepare 1mL of saturated water solution of KI (potassium iodide) 
  add 0.17 mL of saturated water solution of Erythrosine B (at least 95% of purity)  add 0.17 mL of saturated water solution of Erythrosine B (at least 95% of purity)
- add 0.03 mL of 0.004 M KOH (potassium hydroxide) solution in order to achieve pH10  
 </code> </code>
  
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 Evaluation of instrument response functions for lifetime imaging detectors using quenched Rose Bengal solutions\\  Evaluation of instrument response functions for lifetime imaging detectors using quenched Rose Bengal solutions\\ 
 Chemical Physics Letters, Vol.471, p.153-159 (2009)\\  Chemical Physics Letters, Vol.471, p.153-159 (2009)\\ 
-http://dx.doi.org/10.1016/j.cplett.2009.02.001+https://www.sciencedirect.com/science/article/abs/pii/S0009261409001389
  
  
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 Collisional quenching of Erythrosine B as a potential reference dye for impulse response function evaluation\\  Collisional quenching of Erythrosine B as a potential reference dye for impulse response function evaluation\\ 
 Applied Spectroscopy, Vol.63, p.0363-0368 (2009)\\  Applied Spectroscopy, Vol.63, p.0363-0368 (2009)\\ 
-http://www.ingentaconnect.com/content/sas/sas/2009/00000063/00000003/art00017+https://www.osapublishing.org/as/viewmedia.cfm?uri=as-63-3-363&seq=0
  
  
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 Photophysical properties of novel fluorescein derivative and its applications for time-resolved fluorescence spectroscopy\\  Photophysical properties of novel fluorescein derivative and its applications for time-resolved fluorescence spectroscopy\\ 
 Chemical Physics Letters, Vol.493, p.399-403 (2010)\\  Chemical Physics Letters, Vol.493, p.399-403 (2010)\\ 
-http://dx.doi.org/10.1016/j.cplett.2010.05.061+https://www.sciencedirect.com/science/article/abs/pii/S0009261410007256
  
  
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 Picosecond fluorescence of intact and dissolved PSI-LHCI crystals\\  Picosecond fluorescence of intact and dissolved PSI-LHCI crystals\\ 
 Biophysical Journal, Vol.95, p.5851-5861 (2008)\\  Biophysical Journal, Vol.95, p.5851-5861 (2008)\\ 
-http://dx.doi.org/10.1529/biophysj.108.140467+https://www.sciencedirect.com/science/article/pii/S0006349508820012
  
  
howto/how_to_measure_the_instrument_response_function_irf.1572860739.txt.gz · Last modified: 2019/11/04 09:45 by buschmann