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 22:54] – [How to Measure the Instrument Response Function (IRF)] 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 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 |
- | 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