Time-Resolved Fluorescence Wiki

symphotime_tips_and_tricks

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

2020/04/03 13:52		current
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		+
		+
	{{tag>Howto Analysis Tutorial SymPhoTime Tips&Tricks}}		{{tag>Howto Analysis Tutorial SymPhoTime Tips&Tricks}}
-	~~TOC~~



	====== SymPhoTime 64 Analysis Tips and Tricks ======		====== SymPhoTime 64 Analysis Tips and Tricks ======
		+
		+	~~TOC~~
		+
	===== Summary =====		===== Summary =====

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 15:58		current
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	{{tag> tutorial howto FRET PIE analysis time-trace SymPhoTime}}		{{tag> tutorial howto FRET PIE analysis time-trace SymPhoTime}}
-	~~TOC~~	+	~~NOTOC~~

	====== Calculate Ratiometric Single Pair FRET Distributions Using PIE-FRET ======		====== Calculate Ratiometric Single Pair FRET Distributions Using PIE-FRET ======
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	This tutorial shows step-by-step, how to calculate a FRET histogram from single [[glossary:fret\|FRET]]pairs detected under single molecule conditions using pulsed interleaved excitation (PIE). The script requires a spectrally resolved fluorescence time trace, in which donor and acceptor have been excited alternately. As a result, the distribution of FRET efficiencies of different molecules is shown which can be used to detect subpopulations.		This tutorial shows step-by-step, how to calculate a FRET histogram from single [[glossary:fret\|FRET]]pairs detected under single molecule conditions using pulsed interleaved excitation (PIE). The script requires a spectrally resolved fluorescence time trace, in which donor and acceptor have been excited alternately. As a result, the distribution of FRET efficiencies of different molecules is shown which can be used to detect subpopulations.
		+
		+	{{ youtube>cSAbgJpZhrY?large }}

	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.

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	Note: Further information about the sample used in this measurement can be found under: J.L. Fiore et. al., Enthalpy-driven RNA-folding: single-molecule thermodynamics of tetraloop-receptor tertiary interaction, Biochemistry (2009), 48(11), 2550-8. ([[http://pubs.acs.org/doi/abs/10.1021/bi8019788]]).		Note: Further information about the sample used in this measurement can be found under: J.L. Fiore et. al., Enthalpy-driven RNA-folding: single-molecule thermodynamics of tetraloop-receptor tertiary interaction, Biochemistry (2009), 48(11), 2550-8. ([[http://pubs.acs.org/doi/abs/10.1021/bi8019788]]).
-

calculate_ratiometric_single_pair_fret_distributions

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

2014/06/18 15:55		current
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	{{tag> tutorial howto time-trace analysis FRET SymPhoTime}}		{{tag> tutorial howto time-trace analysis FRET SymPhoTime}}
-	~~TOC~~	+	~~NOTOC~~

	====== Calculate Ratiometric Single Pair FRET Distributions ======		====== Calculate Ratiometric Single Pair FRET Distributions ======
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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_and_fit_fcs_traces_with_the_fcs_script

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

2014/06/18 09:21		current
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	===== Background Information =====		===== Background Information =====

-	ATTO488 (ATTO-Tec, Germany) is a suited calibration dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection ~500 – 540 nm.\\	+	* ATTO488 (ATTO-Tec, Germany) is a suited calibration dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection ~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 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.

	===== Step-by-Step Tutorial =====		===== Step-by-Step Tutorial =====
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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.

visualizing_dynamics_using_the_multiframe-flim_script

Anonymous (anonymous@undisclosed.example.com) — 2023-02-17T12:41:10+00:00

2021/03/23 10:44		current
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-	{{tag>howto FLIM multi-frame imaging analysis dynamics tutorial LSM_upgrade SPT SymPhoTime}}	+	{{tag>howto FLIM multi-frame imaging analysis dynamics tutorial LSM_upgrade SymPhoTime}}

-	====== Visualizing Dynamics Using the Multi Frame FLIM Analysis (updated for SymPhoTime v2.5) ======	+	====== Visualizing Dynamics Using the Multi Frame FLIM Analysis (updated for SymPhoTime v2.5 and above) ======

	===== Introduction =====		===== Introduction =====

phasor_plot_structure_separation

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

2025/06/16 08:20		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}}
		+
		+	====== Nikon AX: Phasor-Based Structural Separation Using FLIM ======

	Introduction		Introduction

calculate_ratiometric_fret-images

Anonymous (anonymous@undisclosed.example.com) — 2015-02-25T14:00:57+00:00

2014/06/18 09:24		current
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	~~NOTOC~~		~~NOTOC~~

-	====== Calculate ratiometric FRET-Images ======	+	====== Calculate Ratiometric FRET Images ======


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	{{ calculate_ratiometric_fret-images_with_the_fret-image-script_Image_3.png?348 }}		{{ calculate_ratiometric_fret-images_with_the_fret-image-script_Image_3.png?348 }}

-	Note:Comment: the drop down menu can be opened and closed by clicking on the grey button on the left side of the header of the drop down menu: {{grey_button.png}}	+	Note: the drop down menu can be opened and closed by clicking on the grey button on the left side of the header of the drop down menu: {{grey_button.png}}

	* Start the "FRET Image" script by clicking on "Start".		* Start the "FRET Image" script by clicking on "Start".

pattern_matching

Anonymous (anonymous@undisclosed.example.com) — 2014-06-18T13:26:07+00:00

2014/05/06 14:41		current
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-	{{tag> howto imaging FLIM pattern-matching tutorial analysis}}	+	{{tag> howto imaging FLIM pattern-matching tutorial analysis SymPhoTime}}

	~~NOTOC~~		~~NOTOC~~
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	Pattern Matching Analysis by decomposing a recorded image into different user-defined patterns. Display of the calculated data using an RGB false color model. The calculated amplitudes of the first three defined patterns are depicted in three different colors for each pixel.		Pattern Matching Analysis by decomposing a recorded image into different user-defined patterns. Display of the calculated data using an RGB false color model. The calculated amplitudes of the first three defined patterns are depicted in three different colors for each pixel.
		+
		+	{{ youtube>L68IgRbNSLE?large }}

	==== Open an Image ====		==== Open an Image ====

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 08:09		current
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	{{tag> howto tutorial FCS FLCS crosstalk analysis SymPhoTime}}		{{tag> howto tutorial FCS FLCS crosstalk analysis SymPhoTime}}
-	~~TOC~~	+	~~NOTOC~~

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

calculate_fccs_trace_with_the_grouped_fcs_script

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

2014/06/18 09:21		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 =====

flim_fret_calculation_for_multi_exponential_donors

Anonymous (anonymous@undisclosed.example.com) — 2023-11-21T10:33:47+00:00

2020/05/12 12:39		current
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	~~NOTOC~~		~~NOTOC~~

-	====== FLIM FRET Calculation for Multi Exponential Donors ======	+	====== FLIM-FRET Calculation for Multi Exponential Donors ======


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	Note: This Step-by-Step tutorial was written for SymPhoTime version 2.5. There might be slight differences compared to other software versions. For example the way of setting an intensity threshold has changed. In the video the former version is shown.		Note: This Step-by-Step tutorial was written for SymPhoTime version 2.5. There might be slight differences compared to other software versions. For example the way of setting an intensity threshold has changed. In the video the former version is shown.
-	==== Determine the donor only lifetime using the FLIM script ====	+	==== Determine the donor only lifetime using the FLIM analysis ====


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	Response:\\		Response:\\
	The FLIM script is applied to the file ''CENP-labelled_cells_for_FRET.ptu''. Thereby, a new Window opens:		The FLIM script is applied to the file ''CENP-labelled_cells_for_FRET.ptu''. Thereby, a new Window opens:
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_5.png?600 }}	+	{{ :howto:flim-fret-multiexpd_analysis_flim.png \|}}

	Note:\\		Note:\\
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	* As data channels, check only Channel 2, as in this channel the donor emission has been recorded.		* As data channels, check only Channel 2, as in this channel the donor emission has been recorded.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_6.png }}	+	{{ :howto:flim-fret-multiexpd_ch2.png?400 \|}}

	* Press "Calculate Fast FLIM" to update the image.		* Press "Calculate Fast FLIM" to update the image.
	Response:\\		Response:\\
	The image and the graphs are recalculated, using photons only from channel 2.		The image and the graphs are recalculated, using photons only from channel 2.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_7.png?600 }}	+	{{ :writingroom:flim-fret-multiexpd_fastflim.png \|}}

-	* To enhance the lifetime and intensity contrast, set in the image the intensity from 1 count to 30 counts and the lifetime from 0 to 5 ns.	+	* To enhance the lifetime and intensity contrast, set in the image the intensity from 1 count to 30 counts and the lifetime from 0 to 5 ns.
	Response:\\		Response:\\
	The image scales are adapted.		The image scales are adapted.
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	Now four cells are visible, the two nuclei on the left have longer Fast lifetimes then the other two. These are the cells transfected only with the donor constructs.		Now four cells are visible, the two nuclei on the left have longer Fast lifetimes then the other two. These are the cells transfected only with the donor constructs.

-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_8.png }}	+	{{ :howto:flim-fret-multiexpd_enahncedcontrast.png?400 \|}}

	* Keep the mouse pointer over the image and open the context menu by a right mouse click. Select the "Free ROI" tool.		* Keep the mouse pointer over the image and open the context menu by a right mouse click. Select the "Free ROI" tool.
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	For more information about the different ROI selection tools, check the tutorial [[howto:roi_fitting_using_the_flim_script\|ROI fitting using the FLIM script]].		For more information about the different ROI selection tools, check the tutorial [[howto:roi_fitting_using_the_flim_script\|ROI fitting using the FLIM script]].

-	* Draw a ROI around one of the left cells by maintaining the left mouse button activated while circling the selected part of the image. Then release the left mouse button, press "Shift" continuously and cycle the other left nucleus. This way, the donor only cells are marked as ROI 0.	+	* Draw an ROI around one of the left cells by maintaining the left mouse button activated while circling the selected part of the image. Then release the left mouse button, press "Shift" continuously and cycle the other left nucleus. This way, the donor only cells are marked as ROI 0.

	Response:\\		Response:\\
	* Only the selected area is now shown in color.		* Only the selected area is now shown in color.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_10.png }}	+	{{ :howto:flim-fret-multiexpd_roi.png?400 \|}}

	* In the TCSPC window, the photons from ROI 0 are added in grey.		* In the TCSPC window, the photons from ROI 0 are added in grey.
	{{ flim-fret-calculation_for_multi-exponential_donors_Image_11.png?600 }}		{{ flim-fret-calculation_for_multi-exponential_donors_Image_11.png?600 }}
		+

	* In the decay form on the lower left, select n-exponential reconvolution as fitting model.		* In the decay form on the lower left, select n-exponential reconvolution as fitting model.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_12.png }}	+	{{ :howto:flim-fret-multiexpd_reconvfit.png?400 \|}}

	Response:\\		Response:\\
	* In the TCSPC window, the IRF is displayed in bright red, the data fitting limits are moved to the border of the TCSPC window.		* In the TCSPC window, the IRF is displayed in bright red, the data fitting limits are moved to the border of the TCSPC window.
	* The new fitting parameters "Shift IRF" and "Bkgr IRF" (=background IRF) appear in the fitting parameter table.		* The new fitting parameters "Shift IRF" and "Bkgr IRF" (=background IRF) appear in the fitting parameter table.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_13.png?600 }}	+	{{ :howto:flim-fret-multiexpd_reconvfit_response.png \|}}

	Note:\\		Note:\\
-	The software offers the possibility to fit the data using a n-exponential tailfit or a n-exponential reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. Still a reconvolution fit is usually preferable, especially for FRET determinations, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary, and the short component can be underestimated.	+	The software offers the possibility to fit the data using a n-exponential tailfit, a n-exponential reconvolution fit or a rapid reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. Still a reconvolution fit is usually preferable, especially for FRET determinations, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary, and the short component can be underestimated. The rapid reconvolution model is used for systems with rapid reconvolution electronics (e.g. hybrid PMA-detector + MultiHarp photon counting board), where the peak count rates have been significantly higher than the pile up limit, or in cases, where the repetition rate has been too high to fit the decay completely into the TCSPC-window, e.g. 2PE excitation with 80MHz and dyes with a lifetime above ~3ns.
	For explanation on the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.		For explanation on the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.

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	Response:\\		Response:\\
	The TCSPC curve of ROI 0 is highlighted in green.		The TCSPC curve of ROI 0 is highlighted in green.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_14.png?600 }}	+	{{ :howto:flim-fret-multiexpd_roi-graph.png \|}}

	* At the IRF, click on the "Import" button.		* At the IRF, click on the "Import" button.
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	Response:\\		Response:\\
	The imported IRF is highlighted in red.		The imported IRF is highlighted in red.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_18.png?600 }}	+	{{ :howto:flim-fret-multiexpd_irf-graph.png \|}}

	* As model parameters, select n="2".		* As model parameters, select n="2".
		+	{{ :howto:flim-fret-multiexpd_n2_bearb.png?400 \|}}

	Note:\\		Note:\\
	In this case, the decay is double exponential. As it is not the scope of the tutorial to explain standard lifetime fitting (see the tutorial "ROI fitting using the FLIM script"), we skip the steps of finding the correct fitting model here.		In this case, the decay is double exponential. As it is not the scope of the tutorial to explain standard lifetime fitting (see the tutorial "ROI fitting using the FLIM script"), we skip the steps of finding the correct fitting model here.
-
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_19.png }}

	* Click on "Initial Fit".		* Click on "Initial Fit".
	Response:The fit is calculated.		Response:The fit is calculated.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_20.png?600 }}	+	{{ :howto:flim-fret-multiexpd_initialfit.png \|}}

-	* The software calculates the amplitude weighted average lifetime τ<sub>Av. Amp.</sub>of 2.675. This is the result of the average donor lifetime in absence of FRET.	+	* The software calculates the amplitude weighted average lifetime τ<sub>Av. Amp.</sub>of 2.66. This is the result of the average donor lifetime in absence of FRET.

	Note:\\		Note:\\
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	* Save the result by clicking on "Save Result".		* Save the result by clicking on "Save Result".
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_21.png }}	+
		+	{{ :howto:flim-fret-multiexpd_saveresult_bearb.png?400 \|}}

	Response:\\		Response:\\
	A result file (''FLIM.pqres'') is stored under the raw data file (''CENP-labelled_cells_for_FRET.ptu'').		A result file (''FLIM.pqres'') is stored under the raw data file (''CENP-labelled_cells_for_FRET.ptu'').
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_22.png }}	+	{{ :howto:flim-fret-multiexpd_pqres.png?400 \|}}

	* Now the first part is finished and the FLIM window should be closed.		* Now the first part is finished and the FLIM window should be closed.

flim-fret_calculation_for_single_exponential_donors

Anonymous (anonymous@undisclosed.example.com) — 2020-05-06T09:38:57+00:00

2020/05/05 16:06		current
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	* In this example set the lower intensity threshold to 500 counts.		* In this example set the lower intensity threshold to 500 counts.
		+	{{ :howto:flim-fret-1expd_lowerthresh_500_bearb.png?400 \|}}
	* Click "OK"		* Click "OK"

	Response: In the image, only pixels with higher photon counts are highlighted. Adapt the intensity maximum to see the highlighted pixels more clearly.		Response: In the image, only pixels with higher photon counts are highlighted. Adapt the intensity maximum to see the highlighted pixels more clearly.
-	{{ :howto:flim-fret-1expd_roi_from_thresh_2_v2.png?400 \|}}	+	{{ :howto:flim-fret-1expd_roi_from_thresh_2_v3.png?400 \|}}

	* In the lifetime fitting panel, keep the default Fitting model "Mono-Exp. Donor".		* In the lifetime fitting panel, keep the default Fitting model "Mono-Exp. Donor".

phasor_analysis

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

2018/01/24 14:50		current
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-	{{tag> FLIM howto phasor bin-export analysis tutorial SymPhoTime Globals software}}	+	{{tag> FLIM howto phasor bin-export bin_export analysis tutorial SymPhoTime Globals software}}

	~~TOC~~		~~TOC~~

start

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

2024/12/19 15:56		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}}
	===== SymPhoTime64 / Analysis =====		===== SymPhoTime64 / Analysis =====

calibrate_the_confocal_volume_for_fcs_using_the_fcs_calibration_script

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

2018/09/14 14:34		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 [[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 [[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).\\
	FCS is a technique applicable over a wide range of concentrations, but ideal FCS concentrations are usually between 1 and 20 nM.		FCS is a technique applicable over a wide range of concentrations, but ideal FCS concentrations are usually between 1 and 20 nM.
-
	===== Step-by-Step Tutorial =====		===== Step-by-Step Tutorial =====

how_to_work_with_the_instrument_response_function_irf

Anonymous (anonymous@undisclosed.example.com) — 2023-02-17T12:41:49+00:00

2020/10/22 08:43		current
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-	{{tag>howto IRF tutorial analysis MT LSM_upgrade}}	+	{{tag>howto IRF tutorial analysis MicroTime LSM_upgrade}}


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-	1. First the measured IRF needs to be made accessible for being used in the analysis. Therefore, highlight the file with the measured IRF (single mouse click on the .ptu-file)\\	+	1. First the measured IRF needs to be made accessible for being used in the analysis. Therefore, highlight the file with the measured IRF (single mouse click on the .ptu-file)\\ An example measured IRF can be found in Tutorials workspace [[https://figshare.com/s/4957fcfa684daef86c23]].\\
	2. In Analysis tab, under TCSPC, find `TCSPC Decay`. Click Start;		2. In Analysis tab, under TCSPC, find `TCSPC Decay`. Click Start;

lifetime-fitting_using_the_rapid_reconvolution_algorithm

Anonymous (anonymous@undisclosed.example.com) — 2023-02-17T12:40:42+00:00

2021/03/23 10:39		current
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-		+	{{tag>howto tutorial FLIM analysis SymPhoTime MT LSM_upgrade reconvolution fitting}}
-	{{tag>howto tutorial FLIM analysis SPT SymPhoTime MT LSM_upgrade reconvolution fitting}}	+


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	This tutorial shows step-by-step, how to use the Multi-Exponential Reconvolution fitting model with correction for rapidFLIM. This new approach allows better fitting for data acquired with rapidFLIM capable hardware with count-rates way beyond classical pile up limit.		This tutorial shows step-by-step, how to use the Multi-Exponential Reconvolution fitting model with correction for rapidFLIM. This new approach allows better fitting for data acquired with rapidFLIM capable hardware with count-rates way beyond classical pile up limit.

-	For the principle of rapidFLIM - recording, check the technical note available here: [[https://www.picoquant.com/images/uploads/page/files/16651/2016_soh_appnote_rapidflim.pdf\|Application Note: rapidFLIM]]	+	For the principle of rapidFLIM - recording, check the application note available here:
		+	[[https://www.picoquant.com/dl_technotes/AppNote_rapidFLIM_HiRes.pdf\|Application Note: rapidFLIM]].

	Here in detail the FLIM analysis of SymPhoTime 64 is used to perform a lifetime fit and how to extract and read the results. A mixture of Dragon Green and Nile Red fluorescent microspheres were imaged with more than three frames per second. Tutorials workspace can be downloaded from [[https://figshare.com/s/4957fcfa684daef86c23]].		Here in detail the FLIM analysis of SymPhoTime 64 is used to perform a lifetime fit and how to extract and read the results. A mixture of Dragon Green and Nile Red fluorescent microspheres were imaged with more than three frames per second. Tutorials workspace can be downloaded from [[https://figshare.com/s/4957fcfa684daef86c23]].

roi_fitting_using_the_flim_script

Anonymous (anonymous@undisclosed.example.com) — 2023-02-23T07:08:46+00:00

2015/02/25 13:58		current
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	~~NOTOC~~		~~NOTOC~~

-	====== FLIM ROI Fitting ======	+	====== ROI Fitting Using the FLIM Analysis ======


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-	This tutorial shows step-by-step, how the [[glossary:flim\|FLIM]] script of [[products:SymPhoTime64\|SymPhoTime 64]] can be used to fit several regions of interest (ROIs), and how to extract and interpret the results. In detail, the FLIM script is started using a daisy pollen image from the "Samples" – workspace, then three regions of interest are defined which are finally fitted.	+	This tutorial shows step-by-step, how the [[glossary:flim\|FLIM]] script of [[products:SymPhoTime64\|SymPhoTime 64]] can be used to fit several regions of interest (ROIs), and how to extract and interpret the results. In details, the FLIM script is started using a daisy pollen image from the "Samples" – workspace, then three regions of interest are defined which are finally fitted.

	{{ youtube>g6uyvLQW4nY?large }}		{{ youtube>g6uyvLQW4nY?large }}

separation_of_2_species_with

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

2014/05/23 07:45		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 the FLCS Analysis ======	+	====== 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 =====

-
-	Note:\\
	The two fluorophores ATTO655 (ATTO-Tec, Germany) and Cy5 (GE Healthcare) have almost identical spectral properties and are thus excited with the same wavelength (e.g. 635 nm) and detected at the same spectral range. As also their molecular weight is comparable, the diffusion constant is too similar to distinguish between both dyes in a mixture by means of standard FCS.\\		The two fluorophores ATTO655 (ATTO-Tec, Germany) and Cy5 (GE Healthcare) have almost identical spectral properties and are thus excited with the same wavelength (e.g. 635 nm) and detected at the same spectral range. As also their molecular weight is comparable, the diffusion constant is too similar to distinguish between both dyes in a mixture by means of standard FCS.\\
	Nevertheless, the autocorrelation curves of both dyes differ in the microsecond range. ATTO655 shows practically no photophysical fluctuations. Therefore, FCS curves of ATTO655 can be fitted with the simplest 3D diffusion model, making it very suited for calibration. Cy5 on the contrary, exhibits a distinct bunching term on its FCS trace originating from photophysical processes, i.e. cis/trans isomerization.\\		Nevertheless, the autocorrelation curves of both dyes differ in the microsecond range. ATTO655 shows practically no photophysical fluctuations. Therefore, FCS curves of ATTO655 can be fitted with the simplest 3D diffusion model, making it very suited for calibration. Cy5 on the contrary, exhibits a distinct bunching term on its FCS trace originating from photophysical processes, i.e. cis/trans isomerization.\\
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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]].
-

using_the_antibunching_script

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

2014/05/23 07:39		current
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	{{tag> antibunching howto tutorial correlation analysis SymPhoTime}}		{{tag> antibunching howto tutorial correlation analysis SymPhoTime}}
-	~~TOC~~	+	~~NOTOC~~

-	====== Using the 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.
-

visualizing_dynamics_with_the_multi_frame_flim_analysis

Anonymous (anonymous@undisclosed.example.com) — 2020-05-13T09:39:06+00:00

2015/11/03 14:32		current
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	~~NOTOC~~		~~NOTOC~~

-	====== Visualizing Dynamics with the Multi Frame FLIM Analysis ======	+	====== Visualizing Dynamics Using the Multi Frame FLIM Analysis ======

	===== Summary =====		===== Summary =====
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	- Upper right: Fluorescence Lifetime Histogram. Here the frequency of photon counts corresponding to the individual mean lifetimes is plotted. The settings of the plot can be adapted using the controls on the right of the plot.\\		- Upper right: Fluorescence Lifetime Histogram. Here the frequency of photon counts corresponding to the individual mean lifetimes is plotted. The settings of the plot can be adapted using the controls on the right of the plot.\\
	- Lower left: Lifetime fitting parameters allowing to define the lifetime fitting models. Fitting is then applied to the lifetime graph(s) in the [[glossary:TCSPC]] histogram.\\		- Lower left: Lifetime fitting parameters allowing to define the lifetime fitting models. Fitting is then applied to the lifetime graph(s) in the [[glossary:TCSPC]] histogram.\\
-	- Lower center/left: TCSPC histogram of all photons in the image in green (= dataset 0) and the [[glossary:TCSPC]] histogram of a single pixel in grey (= dataset 1). In red an estimation of the instrument response function (= IRF) is shown. The IRF reconstruction is deducted from the rising edge of the TCSPC histogram.	+	- Lower center/left: [[glossary:TCSPC]] histogram of all photons in the image in green (= dataset 0) and the [[glossary:TCSPC]] histogram of a single pixel in grey (= dataset 1). In red an estimation of the instrument response function (= [[glossary:IRF]]) is shown. The [[glossary:IRF]] reconstruction is deducted from the rising edge of the [[glossary:TCSPC]] histogram.

	* To enhance the lifetime contrast within the image, adapt the color scale from 0 to 15 ns by typing these values as limits for the Fast Lifetime color scale. Also adjust the intensity scale to 1 – 200 counts.		* To enhance the lifetime contrast within the image, adapt the color scale from 0 to 15 ns by typing these values as limits for the Fast Lifetime color scale. Also adjust the intensity scale to 1 – 200 counts.