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.

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).\\

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 =====

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]].
-

burst_duration_and_fcs_diffusion_time

Anonymous (anonymous@undisclosed.example.com) — 2026-01-12T14:58:02+00:00

2026/01/12 15:56		current
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	Gopich, I. V.; Szabo, A. Journal of Chemical Physics 2005, 122, 014707.		Gopich, I. V.; Szabo, A. Journal of Chemical Physics 2005, 122, 014707.

-	Ruttinger, S. Dissertation, 2006.	+	Ruettinger, S. Dissertation, 2006.

-	Dorr, D. Dissertation, 2014.	+	Doerr, D. Dissertation, 2014.

2ffcs

Anonymous (anonymous@undisclosed.example.com) — 2015-11-04T09:41:00+00:00

2014/04/09 22:02		current
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	Correlation analysis that is applied to the fluctuations of the fluorescence intensity. The cross-correlation between the occurrence of events in two different detection volumes is used to introduce an absolute diffusion length into the analysis.		Correlation analysis that is applied to the fluctuations of the fluorescence intensity. The cross-correlation between the occurrence of events in two different detection volumes is used to introduce an absolute diffusion length into the analysis.
		+
		+	see [[glossary:FCS]] for a introduction on FCS.

	===== Experimental Setup =====		===== Experimental Setup =====

symphotime_tips_and_tricks

Anonymous (anonymous@undisclosed.example.com) — 2020-04-03T14:54:26+00:00

2020/04/03 16:54		current
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-	~~TOC~~

	{{tag>Howto Analysis Tutorial SymPhoTime Tips&Tricks}}		{{tag>Howto Analysis Tutorial SymPhoTime Tips&Tricks}}
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	====== SymPhoTime 64 Analysis Tips and Tricks ======		====== SymPhoTime 64 Analysis Tips and Tricks ======
		+
		+	~~TOC~~
		+
	===== Summary =====		===== Summary =====

using_the_flcs_script_for_spectral_crosstalk_removal_via_flccs

Anonymous (anonymous@undisclosed.example.com) — 2015-02-25T14:00:13+00:00

2014/06/19 10:09		current
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	~~NOTOC~~		~~NOTOC~~

-	====== Spectral crosstalk removal via FLCCS ======	+	====== Spectral Crosstalk Removal via FLCCS ======

phasor_analysis

Anonymous (anonymous@undisclosed.example.com) — 2019-08-28T16:15:15+00:00

2019/08/28 18:15		current
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-	{{tag> FLIM howto phasor bin-export binexport analysis tutorial SymPhoTime Globals software}}	+	{{tag> FLIM howto phasor bin-export bin_export analysis tutorial SymPhoTime Globals software}}

	~~TOC~~		~~TOC~~

performing_an_fcs_measurement_with_an_olympus_fv1200_upgrade_kit

Anonymous (anonymous@undisclosed.example.com) — 2015-02-25T13:55:57+00:00

2015/02/06 12:58		current
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	{{tag> howto FCS acquisition video FV1200 Olympus LSM_upgrade}}		{{tag> howto FCS acquisition video FV1200 Olympus LSM_upgrade}}
-	====== Performing an FCS measurement with a Olympus FV1200 upgrade kit======	+	====== Performing a FCS measurement with an Olympus FV1200 Upgrade Kit======

start

Anonymous (anonymous@undisclosed.example.com) — 2026-02-11T14:19:03+00:00

2024/12/19 16:57		current
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	{{topic>howto -FT300 -sample -analysis -draft &nodate&nouser}}		{{topic>howto -FT300 -sample -analysis -draft &nodate&nouser}}

-	===== NovaFLIM / Analysis =====
-
-	{{topic>howto +tutorial +novaflim -draft &nodate&nouser}}

	===== SymPhoTime64 / Analysis =====		===== SymPhoTime64 / Analysis =====

flim_measurement_using_a_nikon_a1_with_a_flim_and_fcs_upgrade

Anonymous (anonymous@undisclosed.example.com) — 2015-06-30T13:07:15+00:00

2015/06/16 13:47		current
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	The general principles of the data acquisition are analogous in all LSM upgrade kits.		The general principles of the data acquisition are analogous in all LSM upgrade kits.
-	The movie was recorded in 02/2014; the recording procedure may be somewhat different in older or newer	+	The movie was recorded in 02/2014 and shows the integration with Nikon A1 system with a NIS version lower than 4.3; the recording procedure may be somewhat different in higher or very old versions.
-	models.	+

	{{ youtube>1Tr9DZ-SkSQ?large }}		{{ youtube>1Tr9DZ-SkSQ?large }}

determination_of_the_focal_width_with_the_focal

Anonymous (anonymous@undisclosed.example.com) — 2015-01-22T14:48:12+00:00

2014/06/18 13:00		current
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	Note: The data in the file is a bead that was excited via pulsed interleaved excitation (PIE) using two perpendicularly polarized lasers, which were guided through a DIC prism. This causes a displacement of the beams, which is used in dual focus FCS (2fFCS) measurements.\\		Note: The data in the file is a bead that was excited via pulsed interleaved excitation (PIE) using two perpendicularly polarized lasers, which were guided through a DIC prism. This causes a displacement of the beams, which is used in dual focus FCS (2fFCS) measurements.\\
-	If data are acquired with a scanner controlled by the SymPhoTime 64 software (i.e. the piezo scanner in the MicroTime 200), the image dimensions are automatically correct. If the image is acquired using an external scanner, the image size has to be defined or transferred before the measurement to make sure that the measured image dimensions are correct. Otherwise, one pixel is considered to a size of 1 µm.	+	If data are acquired with a scanner controlled by the SymPhoTime 64 software (i.e. the piezo scanner in the [[products:MicroTime#MicroTime 200\|MicroTime200]]), the image dimensions are automatically correct. If the image is acquired using an external scanner, the image size has to be defined or transferred before the measurement to make sure that the measured image dimensions are correct. Otherwise, one pixel is considered to a size of 1 µm.

	{{ determination_of_the_focal_width_with_the_focal_Image_6.png }}		{{ determination_of_the_focal_width_with_the_focal_Image_6.png }}

mt200fcs

Anonymous (anonymous@undisclosed.example.com) — 2023-02-17T12:43:50+00:00

2021/06/25 11:07		current
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	======MT200 FCS Measurement ======		======MT200 FCS Measurement ======
-	{{tag>howto mt200 video tutorial}}	+	{{tag>howto MicroTime200 video tutorial}}

	fluorescence correlation spectroscopy (FCS) is a quantitative measurement of the resolution and sensitivity of confocal microscopes. In this tutorial we follow molecular brightness, the count rate per molecule per excitation power, as a well suited parameter for comparison of instrument performance and quality of adjustment. The higher the better: if the system is not optimally aligned, the excitation and/or the detection will be less efficient and the molecular brightness will drop.		fluorescence correlation spectroscopy (FCS) is a quantitative measurement of the resolution and sensitivity of confocal microscopes. In this tutorial we follow molecular brightness, the count rate per molecule per excitation power, as a well suited parameter for comparison of instrument performance and quality of adjustment. The higher the better: if the system is not optimally aligned, the excitation and/or the detection will be less efficient and the molecular brightness will drop.

phasor_plot_structure_separation

Anonymous (anonymous@undisclosed.example.com) — 2025-06-16T08:36:34+00:00

2025/06/16 10:36		current
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	{{tag> FLIM acquisition Nikon AX Phasor video LSM_upgrade howto}}		{{tag> FLIM acquisition Nikon AX Phasor video LSM_upgrade howto}}

-	====== Phasor-Based Structural Separation Using FLIM ======	+	====== Nikon AX: Phasor-Based Structural Separation Using FLIM ======

	Introduction		Introduction

antibunching_measurements

Anonymous (anonymous@undisclosed.example.com) — 2025-04-09T14:01:09+00:00

2025/04/09 16:00		current
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	====== How to Perform Antibunching Measurements ======		====== How to Perform Antibunching Measurements ======

-	Antibunching Measurements are (normally) measured in T2 mode. In T2 mode, all input channels of the TCSPC electronics are treated the same, also the "Sync" channel. Therefore the sync has to be disabled either by disconneting the cable, or by changing the CFD level or trigger level to such a value that the sync is not recognized any more. i.e. Sync rate = 0 . Directly in the Sepia software, the sync out can also be disabled.	+	Antibunching Measurements are (normally) measured in T2 mode. In T2 mode, all input channels of the TCSPC electronics are treated the same, also the "Sync" channel. Therefore the sync has to be disabled either by disconneting the cable, or by changing the CFD level or trigger level to such a value that the sync is not recognized any more. i.e. Sync rate = 0 . Directly in the Sepia software, the sync out can also be disabled.

	The descriptions below only mention physically disconnecting the sync. This is strictly necessary only for the PicoHarp 300 because it only has two channels.		The descriptions below only mention physically disconnecting the sync. This is strictly necessary only for the PicoHarp 300 because it only has two channels.

calculate_ratiometric_single_pair_fret_distributions

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:17:54+00:00

2014/06/18 17:56		current
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	Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\		Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\
	The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\		The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\
-	To record such traces, usually single molecule sensitive detectors as SPAD or Hybrid PMA are necessary to successfully detect single molecule fluorescence.\\	+	To record such traces, usually single molecule sensitive detectors as [[glossary:SPAD]] or [[glossary:Hybrid PMT]]s are necessary to successfully detect single molecule fluorescence.\\

	\\		\\

calculate_ratiometric_single_pair_fret_distributions_using_the_pie-fret_script

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:12:40+00:00

2014/06/18 18:06		current
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	Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\		Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\
	The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\		The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\
-	To record such traces, usually single molecule sensitive detectors as [[glossary:spad\|SPAD]] or Hybrid-PMA are necessary to successfully detect single molecule fluorescence.\\	+	To record such traces, usually single molecule sensitive detectors as [[glossary:spad\|SPAD]] or [[glossary: Hybrid PMT]]s are necessary to successfully detect single molecule fluorescence.\\
	For the pulsed interleaved excitation ([[glossary:pie\|PIE]]) analysis of the script, pulsed excitation using two lasers is required, one laser exciting the donor, the other the acceptor dye. Usually, this is achieved using the multichannel laser driver PDL828 “Sepia II” in combination with two pulsed diode lasers.		For the pulsed interleaved excitation ([[glossary:pie\|PIE]]) analysis of the script, pulsed excitation using two lasers is required, one laser exciting the donor, the other the acceptor dye. Usually, this is achieved using the multichannel laser driver PDL828 “Sepia II” in combination with two pulsed diode lasers.

flim-fret_measurement_using_an_olympus_fv1200_with_a_flim_and_fcs_upgrade

Anonymous (anonymous@undisclosed.example.com) — 2020-05-18T12:53:40+00:00

2015/02/25 14:54		current
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	The general principles of the data acquisition are analogous in all LSM upgrade kits.		The general principles of the data acquisition are analogous in all LSM upgrade kits.
-	The movie was recorded in 02/2014; the recording procedure may be slightly different in older or newer	+	The movie was recorded in 02/2018; the recording procedure may be slightly different in older or newer
	models.		models.

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	{{topic>howto +IRF &nodate&nouser}}		{{topic>howto +IRF &nodate&nouser}}
	{{topic>howto +FLIM +FRET +analysis &nodate&nouser}}		{{topic>howto +FLIM +FRET +analysis &nodate&nouser}}
-	{{topic>howto +FLIM +Nikon}}

intensity_time_trace_analysis

Anonymous (anonymous@undisclosed.example.com) — 2014-06-19T08:12:32+00:00

2014/06/19 10:12		current
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	{{tag> tutorial howto time-trace analysis SymPhoTime}}		{{tag> tutorial howto time-trace analysis SymPhoTime}}

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

	======Intensity Time Trace Analysis======		======Intensity Time Trace Analysis======

using_the_antibunching_script

Anonymous (anonymous@undisclosed.example.com) — 2014-06-19T07:40:32+00:00

2014/06/19 09:36		current
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	{{tag> antibunching howto tutorial correlation analysis SymPhoTime}}		{{tag> antibunching howto tutorial correlation analysis SymPhoTime}}
-	~~TOC~~	+	~~NOTOC~~

	====== Antibunching Analysis ======		====== Antibunching Analysis ======
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	This tutorial shows step-by-step, how to calculate an antibunching curve from a fluorescence time trace of a single emitter, in this example a nanodiamond NV center, excited with a pulsed 530 nm laser.		This tutorial shows step-by-step, how to calculate an antibunching curve from a fluorescence time trace of a single emitter, in this example a nanodiamond NV center, excited with a pulsed 530 nm laser.
		+
		+	{{ youtube>bEyhuWnmFC4?large }}

	===== Background Information =====		===== Background Information =====
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	* Here it is impossible to determine the number of emitters. In this measurement the luminescence stems from several emitters.		* Here it is impossible to determine the number of emitters. In this measurement the luminescence stems from several emitters.
-