howto:how_to_measure_the_instrument_response_function_irf
Differences
This shows you the differences between two versions of the page.
Both sides previous revisionPrevious revisionNext revision | Previous revisionNext revisionBoth sides next revision | ||
howto:how_to_measure_the_instrument_response_function_irf [2016/12/06 11:50] – veiga | howto:how_to_measure_the_instrument_response_function_irf [2021/08/06 07:54] – buschmann | ||
---|---|---|---|
Line 64: | Line 64: | ||
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 | + | |
- | Applied Spectroscopy, Vol.63, p.0363-0368 (2009)\\ | + | ==== Two photon excitation |
- | http://www.ingentaconnect.com/ | + | |
+ | 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, 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 " | ||
+ | |||
+ | |||
+ | ===== How to compensate IRF effects in the analysis | ||
+ | |||
+ | 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: ==== |
Luchowski R., Kapusta P., Szabelski M., Sarkar P., Borejdo J., Gryczynski Z., Gryczynski I.\\ | Luchowski R., Kapusta P., Szabelski M., Sarkar P., Borejdo J., Gryczynski Z., Gryczynski I.\\ | ||
Line 86: | Line 123: | ||
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 92: | Line 129: | ||
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 135: | Line 172: | ||
- | ==== 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: | ||
- | |||
- | ===== 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, | ||
howto/how_to_measure_the_instrument_response_function_irf.txt · Last modified: 2023/09/07 22:55 by peter