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--- howto:how_to_measure_the_instrument_response_function_irf [2016/12/06 12:05] – 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 ====
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  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 78: / Line 81: @@
 ===== 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 =====
@@ Line 113: / 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 119: / 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 125: / 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 135: / 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