Time-Resolved Fluorescence Wiki

calculate_and_fit_fcs_traces_with_the_fcs_script

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:11:17+00:00

2014/06/18 11:22		current
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	* The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see http://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf ]. This list is extensive, but does not claim to be complete.		* The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see http://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf ]. This list is extensive, but does not claim to be complete.
	* Note that the diffusion coefficients of all dyes are temperature dependent.		* Note that the diffusion coefficients of all dyes are temperature dependent.
-	* FCS measurements are usually point measurements acquired with very sensitive detectors (typically SPAD or Hybrid PMT detectors).	+	* FCS measurements are usually point measurements acquired with very sensitive detectors (typically [[glossary:SPAD]] or [[glossary:Hybrid PMT]] detectors).
	* FCS is a technique that can be used over a wide range of concentrations, but ideal FCS concentrations are usually between 1 nM and 20 nM.		* FCS is a technique that can be used over a wide range of concentrations, but ideal FCS concentrations are usually between 1 nM and 20 nM.

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	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_7.png?600 }}		{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_7.png?600 }}

-	Note: A possibility to remove the afterpulsing when working with cw excitation is splitting the light onto two detectors and calculate a cross correlation. Some detectors (mainly Hybrid PMTs) also have so low afterpulsing that the problem does not occur.	+	Note: A possibility to remove the afterpulsing when working with cw excitation is splitting the light onto two detectors and calculate a cross correlation. Some detectors (mainly [[glossary:Hybrid PMT]]s) also have so low afterpulsing that the problem does not occur.

	* If the sample has beenexcited with cw light, we would now save the data by clicking "Save Result" and press afterwards "Transfer to Fit" (both in the "File" dropdown panel). As our sample has been excited with a pulsed laser, we can apply one additional step to increase data quality.		* If the sample has beenexcited with cw light, we would now save the data by clicking "Save Result" and press afterwards "Transfer to Fit" (both in the "File" dropdown panel). As our sample has been excited with a pulsed laser, we can apply one additional step to increase data quality.

calculate_fccs_trace_with_the_grouped_fcs_script

Anonymous (anonymous@undisclosed.example.com) — 2020-04-01T15:05:26+00:00

2015/02/25 15:01		current
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	~~NOTOC~~		~~NOTOC~~

-	====== Calculate Multiple FCCS Traces with the Grouped FCS Script ======	+	====== Calculate FCCS Traces with the Grouped FCS Script ======

	===== Summary =====		===== Summary =====

calibrate_the_confocal_volume_for_fcs_using_the_fcs_calibration_script

Anonymous (anonymous@undisclosed.example.com) — 2019-02-07T12:16:52+00:00

2019/02/07 13:13		current
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	Note: Calibration is a necessary task for FCS measurements. In a calibration procedure, the confocal volume VEff as well as the structural parameter κ (the ratio of z to xy extension) is determined using a reference dye. This is the prerequisite to determine correct diffusion coefficients and concentrations for the sample of interest.\\		Note: Calibration is a necessary task for FCS measurements. In a calibration procedure, the confocal volume VEff as well as the structural parameter κ (the ratio of z to xy extension) is determined using a reference dye. This is the prerequisite to determine correct diffusion coefficients and concentrations for the sample of interest.\\
	ATTO488 (ATTO-Tec, Germany) is a suited dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection around 500 – 540 nm.\\		ATTO488 (ATTO-Tec, Germany) is a suited dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection around 500 – 540 nm.\\
-	The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see [[https://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf]]]. This list is extensive, but does not claim to be complete.\\	+	The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant (see [[https://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf]]). This list is extensive, but does not claim to be complete.\\
	Note that the diffusion coefficients of all dyes are temperature dependent.\\		Note that the diffusion coefficients of all dyes are temperature dependent.\\
	FCS measurements are usually point measurements acquired with very sensitive detectors ([[glossary:spad\|SPAD]] or [[glossary:Hybrid PMT]] detectors).\\		FCS measurements are usually point measurements acquired with very sensitive detectors ([[glossary:spad\|SPAD]] or [[glossary:Hybrid PMT]] detectors).\\

differential_count_rate

Anonymous (anonymous@undisclosed.example.com) — 2017-09-26T11:46:03+00:00

2017/09/24 05:34		current
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	Not really. In case of pulsed signals the average count rate is a misleading quantity. An average count rate value does not take into account when and how those photons are emitted and detected. Interpreting a 100 kcps intensity as a constant emission rate (Poisson mean rate, in math terms) is a misconception. The physics of the measurement is completely different. These photons are obviously not emitted evenly, one by one over the whole one second period. They arrive to the detector bunched, as flashes. These are short time intervals with huge photon density (rate), separated by long "dark", quiet periods.		Not really. In case of pulsed signals the average count rate is a misleading quantity. An average count rate value does not take into account when and how those photons are emitted and detected. Interpreting a 100 kcps intensity as a constant emission rate (Poisson mean rate, in math terms) is a misconception. The physics of the measurement is completely different. These photons are obviously not emitted evenly, one by one over the whole one second period. They arrive to the detector bunched, as flashes. These are short time intervals with huge photon density (rate), separated by long "dark", quiet periods.

-	Getting a final count at "1% of SYNC rate" is a result of **low sampling rate of a high rate signal**.	+	In yet another words, achieving a final count rate at "1% of SYNC rate" is a result of **sparse sampling with dead time**. The sampled signal features much higher photon density, but lasts only for a short time.

	In mathematical terms, "average count rate of 1..2% of SYNC rate" means the overall detection probability, integrated over the whole duration of a measurement. The concept of //differential count rate// is related to the //probability density function//. The detected signal in TCSPC has a very inhomogeneous time distribution.		In mathematical terms, "average count rate of 1..2% of SYNC rate" means the overall detection probability, integrated over the whole duration of a measurement. The concept of //differential count rate// is related to the //probability density function//. The detected signal in TCSPC has a very inhomogeneous time distribution.

	Note: for detectors that exhibit count rate dependent shifting of the IRF, extra care has to be taken when measuring the IRF and directly using it for decay analysis. ((Takuhiro Otosu, Kunihiko Ishii and Tahei Tahara, Note: Simple calibration of the counting-rate dependence of the timing shift of single photon avalanche diodes by photon interval analysis, Rev. Sci. Instrum. 84, 036105 (2013); [[http://dx.doi.org/10.1063/1.4794769]]))		Note: for detectors that exhibit count rate dependent shifting of the IRF, extra care has to be taken when measuring the IRF and directly using it for decay analysis. ((Takuhiro Otosu, Kunihiko Ishii and Tahei Tahara, Note: Simple calibration of the counting-rate dependence of the timing shift of single photon avalanche diodes by photon interval analysis, Rev. Sci. Instrum. 84, 036105 (2013); [[http://dx.doi.org/10.1063/1.4794769]]))

separation_of_2_species_with

Anonymous (anonymous@undisclosed.example.com) — 2014-06-18T12:22:45+00:00

2014/06/18 12:57		current
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	{{tag> howto tutorial FCS FLCS analysis SymPhoTime}}		{{tag> howto tutorial FCS FLCS analysis SymPhoTime}}

-	~~TOC~~	+	~~NOTOC~~

	====== Separation of 2 Species with Different Lifetimes Using FLCS ======		====== Separation of 2 Species with Different Lifetimes Using FLCS ======
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	This tutorial shows step-by-step, how the FLCS analysis can be used to calculate separate autocorrelation curves for the two components of a mixture of ATTO655 and Cy5 based on their different lifetimes.		This tutorial shows step-by-step, how the FLCS analysis can be used to calculate separate autocorrelation curves for the two components of a mixture of ATTO655 and Cy5 based on their different lifetimes.

		+	{{ youtube>xx-WzT5TgqA?large }}

	===== Background Information =====		===== Background Information =====
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	* Click on "Save result" to save this curve. Rename the generated file as "Cy5-FLCS".		* Click on "Save result" to save this curve. Rename the generated file as "Cy5-FLCS".
	* These curves can now be fitted by selecting "Transfer to fit". The process of how to fit a FCS curve is explained in the tutorial [[howto:Calculate and Fit FCS Traces with the FCS Script]].		* These curves can now be fitted by selecting "Transfer to fit". The process of how to fit a FCS curve is explained in the tutorial [[howto:Calculate and Fit FCS Traces with the FCS Script]].
-