howto:how_to_measure_the_instrument_response_function_irf
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howto:how_to_measure_the_instrument_response_function_irf [2016/10/18 11:40] – [Selected literature:] buschmann | howto: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: | + | (For an explanation of the term Instrument Response Function |
+ | |||
+ | The following video shows how to measure na IRF on confocal microscopes. | ||
{{youtube> | {{youtube> | ||
+ | \\ | ||
- | ===== Using scattered excitation light ===== | ||
+ | ==== Low count rate during IRF measurements is important! ==== | ||
- | In case of cuvette based measurement | + | Make sure that the detection count rate is much lower than the count rate during |
- | Note that recording | + | If the IRF should be measured in the UV range on a microscope system with SPAD detectors, |
- | ==== Make sure that the detection count rate is much lower than the count rate used for fluorescence | + | |
+ | ===== Using samples with ultrafast | ||
- | Diluting | + | 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 |
- | 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, | + | ==== General recipe ==== |
+ | * Create a saturated aqueous KI (potassium iodide) solution. (Note, NOT KCl, but iodide.) Beware that KI is pretty well soluble in water. You will need quite a lot of KI. Our experience is that the volume of KI crystals is almost the same as the volume of the water added. The solution MUST be saturated. | ||
+ | |||
+ | * Then add any water soluble fluorescent dye with emission spectrum in the range where you need to record the IRF. | ||
+ | |||
+ | * Good luck with IRF measurements. Do not expect high count rates, but IRF must be recorded at much lower (than fluorescence) count rates anyway. | ||
+ | |||
+ | Our guess is that this quenching trick works generally. Specifically, | ||
+ | |||
+ | Note: the strongly quenched solution will not look fluorescent when watched by eye. However, it must have a strong color (strong absorbtion, high dye concentration). Nevertheless, | ||
+ | |||
+ | Such a cocktail cannot be stored for a long time. Latest the KI will photochemically decompose. | ||
+ | |||
+ | ==== IRF measurement with KI and Erythrosine B ==== | ||
+ | |||
+ | SPAD detectors have wavelengths dependent timing response. IRF recorded at the excitation wavelength may not be useful for precise re-convolution. Using Erythrosine B, the IRF is acquired at the fluorescence wavelength. | ||
+ | |||
+ | Recipe: | ||
+ | |||
+ | < | ||
+ | | ||
+ | add 0.17 mL of saturated water solution of Erythrosine B (at least 95% of purity) | ||
+ | </ | ||
+ | |||
+ | Storage: | ||
+ | |||
+ | < | ||
+ | keep at ~ 4°C, renew the solution after one month | ||
+ | </ | ||
+ | |||
+ | Spectra: | ||
+ | |||
+ | < | ||
+ | excitation from 470 nm to 540 nm | ||
+ | emission from 500 nm to 600 nm | ||
+ | </ | ||
+ | |||
+ | Measurement: | ||
+ | |||
+ | < | ||
+ | Put a droplet on a coverslip, measurement conditions as for fluorescence measurement | ||
+ | </ | ||
- | ===== Using samples with ultrafast decay ===== | ||
- | Some detectors | + | ==== Two photon excitation |
+ | |||
+ | Do not attempt to record an [[glossary: | ||
+ | |||
+ | You can try to excite (by [[glossary: | ||
+ | |||
+ | 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, | ||
+ | |||
+ | ===== Appropriate Count Rate for Measuring | ||
+ | See [[glossary: | ||
+ | |||
+ | ===== How often does the IRF need to be measured? ===== | ||
+ | |||
+ | In spectrometers, | ||
+ | |||
+ | In microscopy-applications, | ||
+ | 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 | ||
+ | |||
+ | |||
+ | ===== 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 | ||
+ | |||
+ | ===== Measuring | ||
+ | |||
+ | We do not recommend to measure | ||
+ | |||
+ | However, in case of cuvette based measurement like in spectrometers, | ||
+ | |||
+ | Note that recording the IRF via scattering requires tuning the emission monochromator to the excitation | ||
==== Selected literature: ==== | ==== Selected literature: ==== | ||
Line 44: | Line 126: | ||
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/ |
Line 50: | Line 132: | ||
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, | Applied Spectroscopy, | ||
- | http://www.ingentaconnect.com/content/sas/ | + | https://www.osapublishing.org/as/viewmedia.cfm? |
Line 56: | Line 138: | ||
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/ |
Line 66: | Line 148: | ||
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/ |
Line 91: | Line 173: | ||
Applied Spectroscopy, | Applied Spectroscopy, | ||
http:// | http:// | ||
- | |||
- | |||
- | ==== General recipe ==== | ||
- | * Create a saturated aqueous KI (potassium iodide) solution. (Note, NOT KCl, but iodide.) Beware that KI is pretty well soluble in water. You will need quite a lot of KI. Our experience is that the volume of KI crystals is almost the same as the volume of the water added. The solution MUST be saturated. | ||
- | * Then add any water soluble fluorescent dye with emission spectrum in the range where you need to record the IRF. | ||
- | |||
- | * Good luck with IRF measurements. Do not expect high count rates, but IRF must be recorded at much lower (than fluorescence) count rates anyway. | ||
- | |||
- | Our guess is that this quenching trick works generally. Specifically, | ||
- | |||
- | Note: the strongly quenched solution will not look fluorescent when watched by eye. However, it must have a strong color (strong absorbtion, high dye concentration). Nevertheless, | ||
- | |||
- | Such a cocktail cannot be stored for a long time. Latest the KI will photochemically decompose. | ||
- | |||
- | ==== IRF measurement with KI and Erythrosine B ==== | ||
- | |||
- | SPAD detectors have wavelengths dependent timing response. IRF recorded at the excitation wavelength may not be useful for precise re-convolution. Using Erythrosine B, the IRF is acquired at the fluorescence wavelength. | ||
- | |||
- | Recipe: | ||
- | |||
- | < | ||
- | | ||
- | 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 | ||
- | </ | ||
- | |||
- | Storage: | ||
- | |||
- | < | ||
- | keep at ~ 4°C, renew the solution after one month | ||
- | </ | ||
- | |||
- | Spectra: | ||
- | |||
- | < | ||
- | excitation from 470 nm to 540 nm | ||
- | emission from 500 nm to 600 nm | ||
- | </ | ||
- | |||
- | Measurement: | ||
- | |||
- | < | ||
- | Put a droplet on a coverslip, measurement conditions as for fluorescence measurement | ||
- | </ | ||
- | | ||
- | Collisional quenching of Erythrosine B as a potential reference dye for impulse response function evaluation\\ | ||
- | Applied Spectroscopy, | ||
- | http:// | ||
- | |||
- | ==== Two photon excitation (TPE) ==== | ||
- | |||
- | Do not attempt to record an [[glossary: | ||
- | |||
- | You can try to excite (by [[glossary: | ||
- | |||
- | 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, | ||
- | |||
- | ===== Appropriate Count Rate for Measuring an IRF ===== | ||
- | See [[glossary: | ||
- | |||
- | ===== 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: |
howto/how_to_measure_the_instrument_response_function_irf.txt · Last modified: 2023/09/07 22:55 by peter