Time-Resolved Fluorescence Wiki

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".

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]]).
-

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]].

lifetime-fitting_using_the_flim_script

Anonymous (anonymous@undisclosed.example.com) — 2023-08-03T14:16:00+00:00

2023/02/17 12:40		current
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-	{{tag>howto tutorial FLIM analysis SymPhoTime MT LSM_upgrade}}	+	{{tag>howto tutorial FLIM analysis SymPhoTime MicroTime LSM_upgrade}}

	====== Lifetime-Fitting Using the FLIM Analysis (updated for SymPhoTime V 2.5 and above) ======		====== Lifetime-Fitting Using the FLIM Analysis (updated for SymPhoTime V 2.5 and above) ======
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	Note:\\		Note:\\
-	The software offers the possibility to fit the data using a n-exponential tailfit, n-exponential reconvolution or rapid reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. A reconvolution fit is usually preferable, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary. A rapidReconvolution fit is needed, if the count rates, with which the image was acquired are significantly above the Pile Up limit. It is also needed, if the pulse frequency is too high for the fluorescence decay to decrease to the background level before the end of the decay window (often, this is the case for 2-Photon-Excitation data acquired with a TiSa-laser with 80MHz repetition rate). Check out the rapidFLIM-analysis tutorial for this case.\\	+	The software offers the possibility to fit the data using a n-exponential tailfit, n-exponential reconvolution or rapid reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. A reconvolution fit is usually preferable, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary. A rapidReconvolution fit is needed, if the count rates, with which the image was acquired are significantly above the Pile Up limit. It is also needed, if the pulse frequency is too high for the fluorescence decay to decrease to the background level before the end of the decay window (often, this is the case for 2-Photon-Excitation data acquired with a TiSa-laser with 80MHz repetition rate). Check out the rapidReconvolution tutorial for this case (https://www.tcspc.com/doku.php/howto:lifetime-fitting_using_the_rapid_reconvolution_algorithm).\\

	For explanations about 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 explanations about 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.\\

lifetime_fitting_using_the_flim_analysis

Anonymous (anonymous@undisclosed.example.com) — 2015-02-25T13:59:23+00:00

2014/06/18 12:19		current
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	{{tag>howto analysis FLIM imaging tutorial SymPhoTime}}		{{tag>howto analysis FLIM imaging tutorial SymPhoTime}}

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

-	====== Lifetime fitting Using the FLIM-analysis ======	+	====== Lifetime Fitting Using the FLIM Analysis ======

update

Anonymous (anonymous@undisclosed.example.com) — 2023-02-23T06:37:29+00:00

2023/02/23 06:15		current
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-	updating...	+	{{tag>howto tutorial FLIM analysis SymPhoTime MT LSM_upgrade}}
		+
		+
		+	====== ROI Fitting Using the FLIM Analysis (updated for SymPhoTime v2.5)======
		+	===== Introduction =====
		+	This tutorial shows step-by-step, how the FLIM script of 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 an image of mouse kidney tissue in the “Tutorials” workspace, [[https://figshare.com/s/4957fcfa684daef86c23]], a suited fitting model is chosen and a pixel-by-pixel fit is performed.\\
		+
		+
		+	===== Step-by-step Tutorial =====
		+
		+	==== Select a file and start the analysis ====
		+
		+	* Start SymPhoTime 64 software (this tutorial uses version 2.5, there might be differences when working with other versions).
		+	* Open the “Tutorials” workspace via File -> Open Workspace from the main menu.\\
		+
		+	Response:\\
		+	The files of the sample workspace are displayed in the workspace panel on the left side of the main window.
		+	* Highlight the file ''Kidney_Cells_FLIM.ptu'' by a single mouse click.
		+	* Select the "Analysis" tab and in there, open the "Imaging" drop-down menu.\\
		+	* Start the FLIM script by clicking on "Start".\\
		+
		+	{{ howto:startflim.png?200 \|}}
		+
		+
		+	Response:\\
		+	The FLIM analysis is applied to the file ''Kidney_Cells_FLIM.ptu''. Thereby, a new Window opens:
		+
		+	{{ howto:kidneyfullscreen3.png \|}}
		+
		+
		+	Note:\\
		+	The window contains five different sections:\\
		+	1: Setting panel\\
		+	Imaging analysis options can be defined in this section.\\
		+	2: [[glossary:Fast FLIM]] image\\
		+	The FLIM preview image displayed in a false color scale. The brightness encodes the intensity, while the color encodes the average "Fast FLIM" lifetime, i.e. the mean arrival times of the photons after the laser pulse. When not defined otherwise, intensity and color scale stretch from minimum to maximum. As the mean arrival time of the background photons in the areas where no fluorescence is detected, is randomly spread over the TCSPC window, the mean photon arrival time of the dark background is usually very long (with an average up to 1/2 of the TCSPC window), which makes the color scale loaded by default sometimes unsuited for displaying the lifetime contrast in the actual sample. In this case, the scale can be adapted.\\
		+	3: 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. The histogram is photon weighted.\\
		+	4: Decay Fitting Parameters\\
		+	Here the lifetime fitting model can be selected. Fitting is then applied to the lifetime graph(s) on the lower center ([[glossary:TCSPC]] histogram).\\
		+	5: [[glossary:TCSPC]] Decay window\\
		+	By default, this set shows the [[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 (=IRF) is shown. The IRF reconstruction is deducted from the rising edge of the [[glossary:TCSPC]] histogram.\\
		+
		+
		+	* To enhance the lifetime contrast, adapt the color scale by typing the limit values for the "Fast Lifetime [ns]" and "Events[cnts]". Additionally set the "Lifetime Histogram" range on the right hand.
		+	* Tick the checkbox of the detection "Data Channel" (i.e. detector), you want to use - in this case detector channel 4 - and press "Calculate FastFLIM".\\
		+
		+	{{ howto:detectionchannel.png?400 \|}}
		+
		+	Response:\\
		+	The FLIM Preview image will get updated. Optimize contrast by adapting "Min" and "Max" values.
		+
		+	==== Select a file and start the analysis ====
		+
		+	* In the FLIM preview image, ROIs can be selected using the context menu (Right Click).\\
		+
		+	{{ howto:selectmagicwand_w.png?300 \|}}
		+
		+	Note:\\
		+
		+	- The following ROI-selection tools are available: Rectangle ROI, Ellipse ROI for simple drawing and Paint ROI for marking the pixels the mouse passed over. The faster the mouse pointer moves, the broader the marked lines will get. Free ROI draws a free shape. The pixels within the drawn shape will be selected. Finally Magic Wand marks area with similar fast lifetime.
		+	- Using keyboard shortcuts ''<CTRL>+<Z>'' you can undo a selection. Redo also with ''<CTRL>+<Shift>+<Z>'' is possible.\\
		+	- You can add several areas to an existing ROI with the same or different selection tool by keeping the ''<Shift>'' key pressed while drawing the additional regions.\\
		+	- If you want to remove some of the pixels from the selected ROI, just keep the ''<CTRL>'' key pressed when using a ROI selection tool to exclude the unwanted parts.\\
		+	- Additionally you can zoom, move or Invert the ROI. Please see the shortcuts provided for each option in the context menu.\\
		+	- If you want to start again from scratch select "Select all as ROI".\\
		+
		+	* In this step, as an example, we mark an ROI with similar lifetime range using the "Magic Wand" tool. The sensitivity of the tool to lifetime differences can be adjusted in a box below the intensity and color scale options.\\
		+
		+	{{ howto:magicwand_w_thresh.png?300 \|}}
		+
		+	* Increase the threshold to 25.\\
		+
		+	{{ howto:inner_magicwand25_3.png \|}}
		+
		+	Response:\\
		+	The parts of the image with a similar fast lifetime range will be selected and the lifetime histogram will get updated accordingly. If you want to combine several spatially separated regions with a similar lifetime into one single ROI, as it's done here, you have to press "Shift" when selecting the spatial separated areas with the Magic wand tool.
		+
		+	Note:\\
		+	Since this function is based on the displayed image, the sensitivity of the selection is dependent on the scaling of the color-encoded component. Changing the color scale (in general the lifetime) can change the sensitivity of this tool.\\
		+
		+
		+	Response:\\
		+	An extra gray decay corresponding to ''ROI 0'' will appear under decay fit in "Decay Fitting" panel.\\
		+
		+
		+	Note:\\
		+	For fine adjustment, you can use any other ROI selection tools from context menu. In this case better to use ''<CTRL>'' or ''<Shift>'' buttons in order to remove pixel with a different or add pixels with similar fast lifetime. Ideally try to select an ROI with 10000 to 100000 counts in peak for getting the optimum statistics. ROIs with lower intensities can also be fitted, but in case of a more-exponential decay, some components might not be resolved.\\
		+
		+
		+	* In order to select an additional ROI, click "New" ROI.\\
		+
		+	{{ howto:addroi.png?400 \|}}
		+
		+
		+	Response:\\
		+	The software assigns sequential numbers to selected ROIs.The whole preview image will appear again.\\
		+
		+	* For the next ROI, first we use the same tool, "Magic Wand" but adapt its threshold to 20.
		+	{{ howto:tubul_magicwand20_3.png \|}}
		+
		+	* Repeat the last two steps in order to select the cell nuclei as the last ROI.\\
		+
		+	{{ howto:nuclei_magicwand30_3.png \|}}
		+
		+	Response:\\
		+	Now you can see fast FLIM (preview) images of "ROI 0", "ROI 1" and "ROI 2" when selecting them in the ROI-list In the Region of Interest-Menu, as well as their corresponding decays in TSCPC window in grey when selecting the different decays in the list.
		+
		+
		+	==== Perform an Initial Lifetime Fit on the Selected Regions (ROIs) ====
		+
		+
		+	* In the forth box, "Decay Fitting", from the Fitting Model drop-down list select "n-Exponential Reconvolution Fit".\\
		+
		+	{{ howto:nexpon_fitmodel.png?400 \|}}
		+
		+	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.\\
		+	The new fitting parameters "Shift IRF" and "Bkgr IRF" (=background IRF) appear in the fitting parameter table.\\
		+
		+	Note:\\
		+	The software offers the possibility to fit the data using an n-exponential tailfit, n-exponential reconvolution or rapid reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. A reconvolution fit is usually preferable, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary. A rapid reconvolution fit is suited, if the image was measured with a very high countrate and a system with a dead time significantly above the pile-up-limit or when the image has been acquired with a repetition rate, which is normally too high for the lifetime of the dye, so that the decay is not at the background level at the end of the decay window. The rapidFLIM-fitting is explained in another tutorial.\\
		+
		+	For explanation about 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.\\
		+
		+
		+	* From "Decay" drop-down list select ''ROI 0'' and click "Initial Fit" (marked in orange).\\
		+	{{ howto:inner_monoexpo_3.png \|}}
		+
		+	Response:\\
		+	A single exponential reconvolution fit is performed. In the TCSPC window, the fit is displayed as a black line. Below, the residuals (= difference between raw data and fit values) are displayed.\\
		+	* Fit values appear in the fitting table. Check the fitting curve, residuals and χ².\\
		+
		+	Note:\\
		+	Usually, a decent fit is characterized by the following criteria:\\
		+	The fitted curve overlays well with the decay curve. In the residual window, the values spread randomly around 0.\\
		+	The χ²-value approaches 1.\\
		+	The calculated fitting values are reasonable.\\
		+	The fitting model with least parameters is preferred.\\
		+
		+
		+	* In this example it is obvious that the applied single exponential reconvolution fit is not suited to fit the decay curve. Increase the "Model Parameters" to n = 2. Again, click on "Initial fit".\\
		+
		+	{{ howto:inner_biexpo_3.png \|}}
		+
		+
		+	Response:\\
		+	The fit quality increases. But if it still doesn't look sufficient. One sees a trend in residuals and not a random distribution around 0. Increase number of "Model Parameters" to three.\\
		+
		+	{{ howto:inner_3expo_3.png \|}}
		+
		+	* Here the fitting model shows a satisfying result. A 3-exponential fitting model should be used for ROI 0.\\
		+
		+	* Try the last two steps with "ROI 1".\\
		+
		+	{{ howto:tubul_3expo_3.png \|}}
		+
		+
		+	* Find the best fit for "ROI 2" in a same manner. It is better always to start from monoexponential and try to find the fitting model with a lower number of components.\\
		+	{{ howto:nuclei_3expo_3.png \|}}
		+
		+	* Three components exponential decay seems to be suitable for all three ROIs. If all ROIs can be fitted with the same model, you can also click on "Fit All" to get all curves fitted with the same model.\\
		+	* Clicking on "Fit All" will fit all active data sets with the same model, using the start parameters of the active data set. If you do not want to fit the complete decay (dataset 1) and the decay of a single pixel (Dataset 2), press "Show All" button:
		+
		+
		+	{{ howto:showall.png \|}}
		+
		+	* Uncheck the first two datasets on top of the parameter table and press "Fit All" again.
		+
		+	{{ howto:deselectdataset1_3.png \|}}
		+
		+
		+
		+
		+	==== Compare the lifetimes and save results ====
		+
		+
		+
		+
		+	* In order to compare the lifetime of these three compartments, apart from fitting parameter table shown above, you can plot them by selecting the "Parameter Plot" on the right side of TCSPC window.\\
		+
		+	Note:\\
		+	The X-Axes is showing the "Dataset Number" corresponding to the dataset listed in Decay drop-down menu (in Dcay Fitting Panel). This means by default "Overall Decay" and "Pixel (0,0)", and then selected ROIs.\\
		+	For the Y-Axes one can select one or multiple fit parameters.\\
		+
		+	* Tick the "tau_Av_Int" checkbox for Y-Axes and zoom in to be able to see the lifetime differences better.\\
		+	{{ howto:parameterplot_3rois_3.png \|}}
		+
		+
		+
		+	* At the end, click on "Save Result" button in parameter panel.\\
		+
		+
		+	{{ howto:saveresultbutton.png?300 \|}}
		+
		+
		+	Response:\\
		+	A result file "FLIM.pqres" is stored under the raw data file ''Kidney_Cell_FLIM.ptu''.\\
		+
		+	* Later, clicking on the result file reopens the file in the same way as it was stored.

roi_fitting_using_the_flim_script

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

2015/02/25 13:58		current
Line 3:		Line 3:
	~~NOTOC~~		~~NOTOC~~

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


Line 9:		Line 9:


-	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 }}

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
Line 1:		Line 1:
-	{{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 =====

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
Line 3:		Line 3:
	~~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.

calculate_ratiometric_fret-images

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

2014/06/18 09:24		current
Line 3:		Line 3:
	~~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".

calculate_ratiometric_single_pair_fret_distributions

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

2014/06/18 15:55		current
Line 1:		Line 1:
	{{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 ======
Line 15:		Line 15:
	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.\\

	\\		\\

separation_of_2_species_with

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

2014/05/23 07:45		current
Line 1:		Line 1:
	{{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 ======


Line 10:		Line 10:

	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.\\
Line 218:		Line 218:
	* 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_anisotropy_image_script

Anonymous (anonymous@undisclosed.example.com) — 2018-04-12T11:44:14+00:00

2018/04/12 10:26		current
Line 51:		Line 51:
	- Lower graph: Anisotropy histogram over the different pixels.		- Lower graph: Anisotropy histogram over the different pixels.

-	Note:	+	Note:
-	The anisotropy values can theoretically spread between -0.5 and 1 for parallel or vertical excitation and emission dipoles respectively. The anisotropy value of 0 is expected in case of very fast rotation or extremely efficient energy transfer processes (e.g. efficient HOMO-FRET). When measuring a randomly aligned ensemble due to photoselection the anisotropy varies with in the limits of -0.2 and 0.4 instead of -0.5 and 1.	+	The anisotropy values can theoretically spread between -0.5 and 1 for perpendicular and parallel excitation and emission dipoles respectively. The anisotropy value of 0 is expected in case of very fast rotation or extremely efficient energy transfer processes (e.g. efficient HOMO-FRET). When measuring a randomly aligned ensemble due to photoselection the anisotropy varies with in the limits of -0.2 and 0.4 instead of -0.5 and 1.

	==== Adapt the script to the image and the system parameters ====		==== Adapt the script to the image and the system parameters ====

lifetime_fitting_using_the_tcpsc_fitting_script

Anonymous (anonymous@undisclosed.example.com) — 2023-09-07T22:43:32+00:00

2014/06/18 16:11		current
Line 9:		Line 9:


-	This tutorial shows step-by-step, how to fit the lifetime of a measured sample. As an example, a single exponential reconvolution fit is used to determine the lifetime of ATTO655 diluted in water.	+	This tutorial shows step-by-step, how to fit a selected model to a decay curve in order to get the lifetime of a measured sample. As an example, a single exponential reconvolution fit is used to determine the lifetime of ATTO655 diluted in water.

	{{ youtube>WupJkKpUVV8?large }}		{{ youtube>WupJkKpUVV8?large }}
Line 50:		Line 50:
	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_6.png?600 }}		{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_6.png?600 }}

-	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, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary.\\	+	Note: The software offers the possibility to use a n-exponential tailfit or a n-exponential reconvolution model fit. A tailfit can be used when the expected lifetimes are significantly longer than the FWHM of instrument response function. Still a reconvolution fit is usually preferable, because the complete decay, including its rising edge is analyzed, while for a tailfit, the start of the fitting range is usually a bit arbitrary.\\
-	For explanation about 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 about the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitted model l equation and the explanation of the different parameters.

	* Click: "Initial Fit" (marked in orange).		* Click: "Initial Fit" (marked in orange).
Line 67:		Line 67:
	The χ²-value approaches 1.\\		The χ²-value approaches 1.\\
	The calculated fitting values are reasonable.\\		The calculated fitting values are reasonable.\\
-	Usually the fitting model with least parameters is selected.\\	+
		+	Usually the fitting model using the smallest amount of adjustable parameters is selected.\\
	=> In this example, the fit is already sufficient.		=> In this example, the fit is already sufficient.

pattern_matching

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

2014/05/06 14:41		current
Line 1:		Line 1:
-	{{tag> howto imaging FLIM pattern-matching tutorial analysis}}	+	{{tag> howto imaging FLIM pattern-matching tutorial analysis SymPhoTime}}

	~~NOTOC~~		~~NOTOC~~
Line 10:		Line 10:

	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 ====

reconvolution_fit

Anonymous (anonymous@undisclosed.example.com) — 2022-08-18T11:31:29+00:00

2022/08/03 15:00		current
Line 8:		Line 8:


-	The last curve in the container, ''crv[1]'', a decay curve in this case, is displayed. The default fitting option is "Exponential Tailfit". We need to select and then assign a curve as an IRF and then change the fitting modality.	+	The last curve in the container, ''crv[1]'', a decay curve in this case, is displayed. The default fitting option is "Exponential Tailfit". In order to perform a reconvolution fit, we need to select and then assign a curve as an IRF and then change the fitting modality.
	{{ :howto:et_fitwindow1.png \|}}		{{ :howto:et_fitwindow1.png \|}}
	\\		\\
Line 14:		Line 14:


-	Clicking on (selecting) ''crv[0]'', the first histogram, the IRF in this particular case, updates the plot. Now, by a right-click on ''crv[0]'' opens a context sensitive dialog, where the "Curve type" can be changed from "Decay" to "IRF".	+	Clicking on (selecting) ''crv[0]'', the first histogram, the IRF in this particular case, updates the plot. Now, a right-click on ''crv[0]'' opens a context sensitive dialog, where the "Curve type" can be changed from "Decay" to "IRF".
	{{ :howto:et_fitwindow2.png \|}}		{{ :howto:et_fitwindow2.png \|}}
	\\		\\

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
Line 1:		Line 1:
	{{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 ======

avoid_pile_up_effect_in_flim_measurements

Anonymous (anonymous@undisclosed.example.com) — 2015-06-03T15:11:25+00:00

2015/03/26 12:09		current
Line 1:		Line 1:

	{{tag> howto FLIM acquisition pile-up TCSPC}}		{{tag> howto FLIM acquisition pile-up TCSPC}}
		+
		+	~~TOC~~

	====== How to Avoid the Pile-up Effect in FLIM Measurements ======		====== How to Avoid the Pile-up Effect in FLIM Measurements ======
Line 39:		Line 41:
	* Determine the number of photons via the data reader (right mouse click) in a very bright pixel within the FastFLIM image		* Determine the number of photons via the data reader (right mouse click) in a very bright pixel within the FastFLIM image
	* Calculate the count rate for this bright pixel: Divide the number of photons per pixel by the exposure time per pixel		* Calculate the count rate for this bright pixel: Divide the number of photons per pixel by the exposure time per pixel
		+
		+	===== Differential Count Rate =====
		+	see [[glossary:Differential Count Rate]]

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
Line 14:		Line 14:
	===== 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 =====
Line 65:		Line 65:
	{{ 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

2014/06/18 09:21		current
Line 2:		Line 2:
	~~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

2018/09/14 14:34		current
Line 17:		Line 17:
	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 =====

data_file_import

Anonymous (anonymous@undisclosed.example.com) — 2022-08-04T13:24:27+00:00

2022/08/03 14:07		current
Line 3:		Line 3:
	Create a new workspace or open an existing one:		Create a new workspace or open an existing one:

-	{{ :howto:et2_createopen.png?370 \| File menu }}	+	{{ :howto:et2_createopen.png \| File menu }}


-	Then select Import Files. A new dialog opens where the file is selected:	+	Then select Import Files. A new dialog opens, where the data file is selected:

-	{{ :howto:et2_fileimport.png?1161 \| Selects the file to be imported }}	+	{{ :howto:et2_fileimport.png \| Selects the file to be imported }}

-	The file selected above is a sample data file, ''Decay_Coumarin_6.phu'' installed during the installation of the native TimeHarp260 software. This file contains two histograms: an Instrument Response Function (IRF) and a decay curve of Coumarin6 dissolved in ethanol. During file import, before pressing the Open button, it is possible to preview and step through the data content of selected file using the control elements on the right hand side of the dialog.	+	The file selected above is a sample data file, ''Decay_Coumarin_6.phu'' installed during the installation of the [[https://www.picoquant.com/dl_software/TimeHarp260/TimeHarp260_SW_and_DLL_V3_2.zip\|native TimeHarp260 software]]. This file contains two histograms: an [[howto:how_to_measure_the_instrument_response_function_irf\|Instrument Response Function (IRF)]] and a decay curve of Coumarin6 dissolved in ethanol. During file import, before pressing the __O__pen button, it is possible to preview and step through the data content of selected file using the control elements on the right hand side of the dialog.

	The same data file, when opened in the native software, looks like this:		The same data file, when opened in the native software, looks like this:

-	{{ :howto:et2_originalhistograms.png?1033 \| Original data file content }}	+	{{ :howto:et2_originalhistograms.png \| Original data file content }}

-	Trace #0 contains the IRF, Trace #2 is the decay curve. The other data blocks are empty. Both Trace#0 and #2 will be imported into a single ''.etc'' data container. This will make the data analysis easier. In general, such a ''.phu'' file may contain many more histograms. It is also possible that an IRF measurement and a decay measurement are stored in two separate ''.phu'' files. This is no problem, but then the file import procedure has to be repeated and the imported curves will end up in dedicated .etc data containers.	+	Trace #0 shows the IRF, Trace #2 is the decay curve. The other data blocks are empty. Both Trace #0 and #2 will be imported into a single ''.etc'' data container. This will make the data analysis easier. In general, such a ''.phu'' file may contain many more histograms.
		+
		+	It is also possible that an IRF measurement and a decay measurement are stored in two separate ''.phu'' files. This is no problem, but then the file import procedure has to be repeated for each file and the imported curves will end up in dedicated ''.etc'' data containers.

	After importing ''Decay_Coumarin_6.phu'', the workspace looks like this:		After importing ''Decay_Coumarin_6.phu'', the workspace looks like this:

-	{{ :howto:et2_workspace1.png?338 \|}}	+	{{ :howto:et2_workspace1.png \|}}
	\\		\\
	\\		\\
	After expanding the data container:		After expanding the data container:
-	{{ :howto:et2_workspace2.png?340 \|}}	+	{{ :howto:et2_workspace2.png \|}}

	Two histograms have been imported. They are shown as "Decay" because EasyTau does not know their assignment yet.		Two histograms have been imported. They are shown as "Decay" because EasyTau does not know their assignment yet.
		+	\\
		+	\\
		+
		+
		+	==== Multiple File Import ====
		+
		+	Importing multiple data files is easy. Use CTRL + Click to select multiple files:
		+
		+	{{ :howto:et2_fileimport2.png \|}}
		+	\\
		+	\\
		+	Each input ''.phu'' file has got a dedicated ''.etc'' data container:
		+	{{ :howto:et2_workspace3.png \|}}
		+	\\
		+	\\
		+	Unlike ''Decay_Coumarin_6.phu'' containing two histograms, both ''Anthracene_EtOH.phu'' and ''IRF375.phu'' contains only one histogram. These two measurements actually belong to each other. In order to perform reconvolution fitting of the Antracene decay, the corresponding IRF measured at 375nm excitation wavelength will be needed.
		+

diamond_nv_centers

Anonymous (anonymous@undisclosed.example.com) — 2018-04-19T12:11:55+00:00

2015/02/25 14:00		current

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
Line 1:		Line 1:
-	{{tag>howto IRF tutorial analysis MT LSM_upgrade}}	+	{{tag>howto IRF tutorial analysis MicroTime LSM_upgrade}}


Line 35:		Line 35:


-	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;

intensity_time_trace_analysis

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

2014/05/07 16:14		current
Line 1:		Line 1:
	{{tag> tutorial howto time-trace analysis SymPhoTime}}		{{tag> tutorial howto time-trace analysis SymPhoTime}}

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

	======Intensity Time Trace Analysis======		======Intensity Time Trace Analysis======
Line 10:		Line 10:

	This tutorial shows step-by-step, how to draw intensity time traces from a single molecule. The script requires a point measurement of an immobilized fluorophore. When applied to images, it can be used to check for line and frame marker presence and assignment.		This tutorial shows step-by-step, how to draw intensity time traces from a single molecule. The script requires a point measurement of an immobilized fluorophore. When applied to images, it can be used to check for line and frame marker presence and assignment.
		+
		+	{{ youtube>D4BBTLBvanQ?large }}
		+

	\\		\\

symphotime_tips_and_tricks

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

2020/04/03 13:52		current
Line 1:		Line 1:
		+
		+
	{{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 =====

using_the_antibunching_script

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

2014/05/23 07:39		current
Line 1:		Line 1:
	{{tag> antibunching howto tutorial correlation analysis SymPhoTime}}		{{tag> antibunching howto tutorial correlation analysis SymPhoTime}}
-	~~TOC~~	+	~~NOTOC~~

-	====== Using the Antibunching Analysis ======	+	====== Antibunching Analysis ======


Line 9:		Line 9:

	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 =====
Line 77:		Line 79:

	* 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.
-