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--- howto:how_to_measure_the_instrument_response_function_irf [2016/12/06 11:50] – veiga
+++ howto:how_to_measure_the_instrument_response_function_irf [2023/09/07 22:55] (current) – [Using samples with ultrafast decay] peter
@@ Line 4: / Line 4: @@
 ====== 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}}
+\\
-==== 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 =====
-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 ====
@@ Line 43: / Line 47: @@
  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 0.03 mL of 0.004 M KOH (potassium hydroxide) solution in order to achieve pH10
 </code>
@@ Line 64: / Line 67: @@
 Put a droplet on a coverslip, measurement conditions as for fluorescence measurement
 </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 Spectroscopy, Vol.63, p.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) 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 often does the IRF need to be measured? =====
+In spectrometers, the IRF is usually measured before or after each measurement.
+In microscopy-applications, this is usually not practical; and often it is sufficient to measure the IRF once during a measurement series, provided that the system has had time to warm up (~15min), and neither the repetition rate nor the intensity at the diode laser driver is changed (the current which drives the laser).
+If the intensity needs to be changed, the optical attenuation can be adapted.
+A special case are systems with 2-Photon-Excitation (2PE). Here, usually TiSa-lasers are used which have fs-pulses, therefore the IRF is normally determined by the detector. In these cases, often the IRF can be measured once (and the excitation wavelength is not important, provided that the IRF is measured with a quenched dye and the same filterset is used as for the sample), and re-used later. Over time or upon changes of the excitation wavelength, the position of the IRF can shift slightly, but this is accounted for with the "IRF-shift"-parameter of the fitting equation.
+===== 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.
-==== 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.\\
@@ Line 86: / Line 126: @@
 Evaluation of instrument response functions for lifetime imaging detectors using quenched Rose Bengal solutions\\
 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
@@ Line 92: / Line 132: @@
 Collisional quenching of Erythrosine B as a potential reference dye for impulse response function evaluation\\
 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
@@ Line 98: / Line 138: @@
 Photophysical properties of novel fluorescein derivative and its applications for time-resolved fluorescence spectroscopy\\
 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
@@ Line 108: / Line 148: @@
 Picosecond fluorescence of intact and dissolved PSI-LHCI crystals\\
 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
@@ Line 135: / Line 175: @@
-==== 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.