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 22:48] – [Make sure that the detection count rate is much lower than the count rate used for fluorescence decay measurement.] 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 ===== | ||
- | In case of cuvette based measurement the simplest procedure | + | === Low count rate during IRF measurements |
- | 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, | + | **Note: |
- | + | ||
- | ==== Make sure that the detection count rate is much lower than the count rate used for fluorescence decay measurement. | + | |
- | + | ||
- | Diluting the scattering solution is better than using grey filters. Ideal is when the decay and the IRF are recorded at the same [[glossary: | + | |
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, | 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, | ||
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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. | ||
- | |||
- | ==== Selected literature: ==== | ||
- | |||
- | Luchowski R., Kapusta P., Szabelski M., Sarkar P., Borejdo J., Gryczynski Z., Gryczynski I.\\ | ||
- | Förster resonance energy transfer (FRET)-based picosecond lifetime reference for instrument response evaluation \\ | ||
- | Measurement Science and Technology, Vol.20, 095601 (2009)\\ | ||
- | http:// | ||
- | |||
- | |||
- | Luchowski R., Gryczynski Z., Sarkar P., Borejdo J., Szabelski M., Kapusta P., Gryczynski I.\\ | ||
- | Instrument response standard in time-resolved fluorescence\\ | ||
- | Review of Scientific Instruments, | ||
- | http:// | ||
- | |||
- | |||
- | Szabelski M., Luchowski R., Gryczynski Z., Kapusta P., Ortmann U., Gryczynski I.\\ | ||
- | Evaluation of instrument response functions for lifetime imaging detectors using quenched Rose Bengal solutions\\ | ||
- | Chemical Physics Letters, Vol.471, p.153-159 (2009)\\ | ||
- | http:// | ||
- | |||
- | |||
- | 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, | ||
- | http:// | ||
- | |||
- | |||
- | Szabelski M., Gryczynski Z., Gryczynski I.\\ | ||
- | Photophysical properties of novel fluorescein derivative and its applications for time-resolved fluorescence spectroscopy\\ | ||
- | Chemical Physics Letters, Vol.493, p.399-403 (2010)\\ | ||
- | http:// | ||
- | |||
- | |||
- | The dye **pinacyanol iodide** has a very fast decay time (in methanol 6 ps) and very broad emission spectrum (starting at around 550nm), thus suitable for determination of the IRF in a very broad range. | ||
- | |||
- | Pinacyanol iodide is also known as Quinaldine blue, 1,1' -Diethyl-2, | ||
- | |||
- | van Oort B., Amunts A., Borst J. W., van Hoek A., Nelson N., van Amerongen H., Croce R.\\ | ||
- | Picosecond fluorescence of intact and dissolved PSI-LHCI crystals\\ | ||
- | Biophysical Journal, Vol.95, p.5851-5861 (2008)\\ | ||
- | http:// | ||
- | |||
- | |||
- | The dye **Allura Red** has also a remarkably short fluorescence lifetime of about 10 ps. This short lifetime does not depend on the emission (observation) wavelength (from 550 nm to 750 nm) and it is another candidate for impulse response measurements. | ||
- | |||
- | Allura Red is Disodium 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonate, | ||
- | |||
- | Rahul Chib, Sunil Shah, Zygmunt Gryczynski, Rafal Fudala, Julian Borejdo, Bogumil Zelent, Maria G Corradini, Richard D Ludescher, Ignacy Gryczynski\\ | ||
- | Standard reference for instrument response function in fluorescence lifetime measurements in visible and near infrared\\ | ||
- | Measurement Science and Technology, Vol.27, 027001 (2016)\\ | ||
- | http:// | ||
- | |||
- | Pyridinium Styryl Dyes have also been used as IRF standard: | ||
- | |||
- | Lei Li, Mengfang Chang, Hua Yi, Menghui Jia, Siaodan Cao, Zhongneng Zhou, Sanjun Zhang, Haifeng Pan, Chun-Wei Shih, Ralph Jimenez, Jianhua Xu\\ | ||
- | Using Pyridinium Styryl Dyes as the Standards of Time-Resolved Instrument Response\\ | ||
- | Applied Spectroscopy, | ||
- | http:// | ||
- | |||
- | Even dye with lifetimes that are usually in the ns range can be used, if the lifetime can be quenched to a few ps. Quenching can be achieved using a saturated iodide solution. For details see: | ||
- | |||
- | Mengwei Liu, Mengui Jia, Haifeng Pan, Lei Li, Mengfang Chang, Hua Ren, Françoise Argoul, Sanjun Zhang, Jianhua Xu\\ | ||
- | Instrument Response Standard in Time-Resolved Fluorescence Spectroscopy at Visible Wavelength: Quenched Fluorescein Sodium\\ | ||
- | Applied Spectroscopy, | ||
- | http:// | ||
- | |||
- | |||
- | |||
==== General recipe ==== | ==== General recipe ==== | ||
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| | ||
add 0.17 mL of saturated water solution of Erythrosine B (at least 95% of purity) | 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 | ||
</ | </ | ||
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Put a droplet on a coverslip, measurement conditions as for fluorescence measurement | Put a droplet on a coverslip, measurement conditions as for fluorescence measurement | ||
</ | </ | ||
- | ((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, | + | |
- | http:// | + | |
==== Two photon excitation (TPE) ==== | ==== Two photon excitation (TPE) ==== | ||
Line 156: | Line 81: | ||
===== Appropriate Count Rate for Measuring an IRF ===== | ===== Appropriate Count Rate for Measuring an IRF ===== | ||
See [[glossary: | 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 ===== | ===== How to compensate IRF effects in the analysis of time domain measurements ===== | ||
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Note: All analysis packages from PicoQuant use the [[glossary: | 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: ==== | ||
+ | |||
+ | Luchowski R., Kapusta P., Szabelski M., Sarkar P., Borejdo J., Gryczynski Z., Gryczynski I.\\ | ||
+ | Förster resonance energy transfer (FRET)-based picosecond lifetime reference for instrument response evaluation \\ | ||
+ | Measurement Science and Technology, Vol.20, 095601 (2009)\\ | ||
+ | http:// | ||
+ | |||
+ | |||
+ | Luchowski R., Gryczynski Z., Sarkar P., Borejdo J., Szabelski M., Kapusta P., Gryczynski I.\\ | ||
+ | Instrument response standard in time-resolved fluorescence\\ | ||
+ | Review of Scientific Instruments, | ||
+ | http:// | ||
+ | |||
+ | |||
+ | Szabelski M., Luchowski R., Gryczynski Z., Kapusta P., Ortmann U., Gryczynski I.\\ | ||
+ | Evaluation of instrument response functions for lifetime imaging detectors using quenched Rose Bengal solutions\\ | ||
+ | Chemical Physics Letters, Vol.471, p.153-159 (2009)\\ | ||
+ | https:// | ||
+ | |||
+ | |||
+ | 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, | ||
+ | https:// | ||
+ | |||
+ | |||
+ | Szabelski M., Gryczynski Z., Gryczynski I.\\ | ||
+ | Photophysical properties of novel fluorescein derivative and its applications for time-resolved fluorescence spectroscopy\\ | ||
+ | Chemical Physics Letters, Vol.493, p.399-403 (2010)\\ | ||
+ | https:// | ||
+ | |||
+ | |||
+ | The dye **pinacyanol iodide** has a very fast decay time (in methanol 6 ps) and very broad emission spectrum (starting at around 550nm), thus suitable for determination of the IRF in a very broad range. | ||
+ | |||
+ | Pinacyanol iodide is also known as Quinaldine blue, 1,1' -Diethyl-2, | ||
+ | |||
+ | van Oort B., Amunts A., Borst J. W., van Hoek A., Nelson N., van Amerongen H., Croce R.\\ | ||
+ | Picosecond fluorescence of intact and dissolved PSI-LHCI crystals\\ | ||
+ | Biophysical Journal, Vol.95, p.5851-5861 (2008)\\ | ||
+ | https:// | ||
+ | |||
+ | |||
+ | The dye **Allura Red** has also a remarkably short fluorescence lifetime of about 10 ps. This short lifetime does not depend on the emission (observation) wavelength (from 550 nm to 750 nm) and it is another candidate for impulse response measurements. | ||
+ | |||
+ | Allura Red is Disodium 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonate, | ||
+ | |||
+ | Rahul Chib, Sunil Shah, Zygmunt Gryczynski, Rafal Fudala, Julian Borejdo, Bogumil Zelent, Maria G Corradini, Richard D Ludescher, Ignacy Gryczynski\\ | ||
+ | Standard reference for instrument response function in fluorescence lifetime measurements in visible and near infrared\\ | ||
+ | Measurement Science and Technology, Vol.27, 027001 (2016)\\ | ||
+ | http:// | ||
+ | |||
+ | Pyridinium Styryl Dyes have also been used as IRF standard: | ||
+ | |||
+ | Lei Li, Mengfang Chang, Hua Yi, Menghui Jia, Siaodan Cao, Zhongneng Zhou, Sanjun Zhang, Haifeng Pan, Chun-Wei Shih, Ralph Jimenez, Jianhua Xu\\ | ||
+ | Using Pyridinium Styryl Dyes as the Standards of Time-Resolved Instrument Response\\ | ||
+ | Applied Spectroscopy, | ||
+ | http:// | ||
+ | |||
+ | Even dye with lifetimes that are usually in the ns range can be used, if the lifetime can be quenched to a few ps. Quenching can be achieved using a saturated iodide solution. For details see: | ||
+ | |||
+ | Mengwei Liu, Mengui Jia, Haifeng Pan, Lei Li, Mengfang Chang, Hua Ren, Françoise Argoul, Sanjun Zhang, Jianhua Xu\\ | ||
+ | Instrument Response Standard in Time-Resolved Fluorescence Spectroscopy at Visible Wavelength: Quenched Fluorescein Sodium\\ | ||
+ | Applied Spectroscopy, | ||
+ | http:// | ||
+ | |||
+ | |||
+ | |||
+ |
howto/how_to_measure_the_instrument_response_function_irf.txt · Last modified: 2023/09/07 22:55 by peter