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howto:how_to_measure_the_instrument_response_function_irf [2016/12/06 12:50]
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howto:how_to_measure_the_instrument_response_function_irf [2016/12/06 13:05]
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 Put a droplet on a coverslip, measurement conditions as for fluorescence measurement ​ Put a droplet on a coverslip, measurement conditions as for fluorescence measurement ​
 </​code>​ </​code>​
- ((Szabelski M., Iliev D., Sarkar P., Luchowski R., Gryczynski Z., Kapusta P., Erdmann R., Gryczinski I.\\  +  
-Collisional quenching of Erythrosine B as a potential reference dye for impulse response function evaluation\\ ​ + 
-Applied SpectroscopyVol.63p.0363-0368 (2009)\\  +==== Two photon excitation ​(TPE) ==== 
-http://www.ingentaconnect.com/​content/​sas/​sas/​2009/​00000063/​00000003/​art00017)+ 
 +Do not attempt to record an [[glossary:​IRF]] at the fundamental ​(IR) wavelengthThe resulting pulse form would be meaningless anyway. 
 + 
 +You can try to excite (by [[glossary:​TPE]]of course) any of the above mentioned fast decay time fluorophores and record their responseThe signal will be wery weakbut this is not a problem for IRF measurements. 
 + 
 +With microscopes it is convenient to record the second harmonic signal that is generated on the surface of urea crystalsThe best is to let evaporate a droplet of concentrated urea solution on a clear cover slipThe resulting film of micro-crystals is easy to target. 
 + 
 +Ureaaka Carbamide or Carbonyldiamide,​ CAS Number:​57-13-6 
 + 
 +===== Appropriate Count Rate for Measuring an IRF ===== 
 +See [[glossary:​Differential Count Rate]] 
 +  
 +===== How to compensate IRF effects in the analysis of time domain measurements ===== 
 + 
 +There are two major ways of compensating IRF effects: 
 + 
 +    *correct the effects in the data (**de**convolution) 
 +    *take the effects into account in your model equation (**re**convolution)  
 + 
 +Note: All analysis packages from PicoQuant use the [[glossary:​reconvolution]] methodThe reason is simple: [[glossary:​Deconvolution]] has many disadvantages:​ It is rather slowit is an ill posed problem producing a lot of noise (at best), it does not get you any nearer to the model parameters, and it is very likely to produce artefacts without allowing to tell apart artefacts and effects
 + 
 +===== Measuring the IRF as scattered excitation light ===== 
 + 
 +We do not recommend to measure the IRF as scatters light in microscopy, due to the color dependence of SPAD detectors, which are generally used in microscopy. Furthermore,​ even with detectors lacking color effects, it is disadvantageous,​ due to the many reflection peaks found along the light pathway in microscope set up.  
 + 
 +Howeverin case of cuvette based measurement like in spectrometers,​ this is  the method of choiceThe simplest procedure is to use a very diluted solution of colloidal silica. (LUDOX is often usedLUDOX is a trade mark by DuPontIt can be purchased via Aldrich or Sigma.) Do not use "non-diary cafe creamer"​ or glycogen, mentioned in older literature. These compunds are fluorescent. The scattering solution must be really weak, typical starting "​concentration"​ is one droplet of the colloid from the original LUDOX bottle ​(as delivered with you systemdiluted 100x. If the signal is too strong, dilute further. 
 + 
 +Note that recording the IRF via scattering requires tuning the emission monochromator to the excitation wavelengthIn filter based machines, e.g. FluoTime100 this means removing the emission bandpass or longpass filter. In microscopes,​ this is equivalent to replacing the detection filter with an OD filter (OD = optical density, grey filterand recording the back scattering from e.g. a clean glass cover slip. Beware, on some confocal LSMs it is simply not possible to measure at the excitation wl. due to the restrictions introduced into the system software by the manufacturer. In case of measurements of solid samples in FT100 or FT200 spectrometers,​ the surface scattering is usually so strong that it is not necessary to mount a special sample for IRF. Tuning the mono to the exc. wl. and attenuating the excitation beam is sufficient. ​
  
-==== Selected literature ​to masure IRF as an ultrafast decay: ====+==== Selected literature: ====
  
 Luchowski R., Kapusta P., Szabelski M., Sarkar P., Borejdo J., Gryczynski Z., Gryczynski I.\\  Luchowski R., Kapusta P., Szabelski M., Sarkar P., Borejdo J., Gryczynski Z., Gryczynski I.\\ 
Line 135: Line 162:
    
  
-==== Two photon excitation (TPE) ==== 
- 
-Do not attempt to record an [[glossary:​IRF]] at the fundamental (IR) wavelength. The resulting pulse form would be meaningless anyway. 
- 
-You can try to excite (by [[glossary:​TPE]],​ of course) any of the above mentioned fast decay time fluorophores and record their response. The signal will be wery weak, but this is not a problem for IRF measurements. 
- 
-With microscopes it is convenient to record the second harmonic signal that is generated on the surface of urea crystals. The best is to let evaporate a droplet of concentrated urea solution on a clear cover slip. The resulting film of micro-crystals is easy to target. 
- 
-Urea, aka Carbamide or Carbonyldiamide,​ CAS Number:​57-13-6 
- 
-===== Appropriate Count Rate for Measuring an IRF ===== 
-See [[glossary:​Differential Count Rate]] 
-  
-===== How to compensate IRF effects in the analysis of time domain measurements ===== 
- 
-There are two major ways of compensating IRF effects: 
- 
-    *correct the effects in the data (**de**convolution) 
-    *take the effects into account in your model equation (**re**convolution) ​ 
- 
-Note: All analysis packages from PicoQuant use the [[glossary:​reconvolution]] method. The reason is simple: [[glossary:​Deconvolution]] has many disadvantages:​ It is rather slow, it is an ill posed problem producing a lot of noise (at best), it does not get you any nearer to the model parameters, and it is very likely to produce artefacts without allowing to tell apart artefacts and effects. 
- 
-===== Measuring the IRF as scattered excitation light ===== 
- 
-We do not recommend to measure the IRF as scatters light in microscopy, due to the color dependence of SPAD detectors, which are generally used in microscopy. Furthermore,​ even with detectors lacking color effects, it is disadvantageous,​ due to the many reflection peaks found along the light pathway in a microscope set up.  
- 
-However, in case of cuvette based measurement like in spectrometers,​ this is  the method of choice. The simplest procedure is to use a very diluted solution of colloidal silica. (LUDOX is often used, LUDOX is a trade mark by DuPont. It can be purchased via Aldrich or Sigma.) Do not use "​non-diary cafe creamer"​ or glycogen, mentioned in older literature. These compunds are fluorescent. The scattering solution must be really weak, typical starting "​concentration"​ is one droplet of the colloid from the original LUDOX bottle (as delivered with you system) diluted 100x. If the signal is too strong, dilute further. 
- 
-Note that recording the IRF via scattering requires tuning the emission monochromator to the excitation wavelength. In filter based machines, e.g. FluoTime100 this means removing the emission bandpass or longpass filter. In microscopes,​ this is equivalent to replacing the detection filter with an OD filter (OD = optical density, grey filter) and recording the back scattering from e.g. a clean glass cover slip. Beware, on some confocal LSMs it is simply not possible to measure at the excitation wl. due to the restrictions introduced into the system software by the manufacturer. In case of measurements of solid samples in FT100 or FT200 spectrometers,​ the surface scattering is usually so strong that it is not necessary to mount a special sample for IRF. Tuning the mono to the exc. wl. and attenuating the excitation beam is sufficient. ​ 
  
  
howto/how_to_measure_the_instrument_response_function_irf.txt · Last modified: 2019/11/04 10:45 by buschmann