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 [2019/11/04 09:45] – [Appropriate Count Rate for Measuring an IRF] buschmann | ||
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{{youtube> | {{youtube> | ||
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- | ===== Using scattered excitation light ===== | ||
- | |||
- | |||
- | In case of cuvette based measurement 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 " | ||
- | |||
- | 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, | ||
==== Make sure that the detection count rate is much lower than the count rate used for fluorescence decay measurement. ==== | ==== Make sure that the detection count rate is much lower than the count rate used for fluorescence decay measurement. ==== | ||
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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 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. | ||
+ | |||
+ | ==== 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 | ||
+ | </ | ||
+ | |||
+ | |||
+ | ==== 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 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 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 " | ||
+ | |||
+ | |||
+ | ===== 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: | ||
+ | |||
+ | ===== 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, | ||
+ | |||
+ | 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 wavelength. In filter based machines, e.g. FluoTime100 this means removing the emission bandpass or longpass filter. In microscopes, | ||
==== Selected literature: ==== | ==== Selected literature: ==== | ||
Line 91: | Line 170: | ||
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