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.

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
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-	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 }}
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	{{ 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).
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	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.

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.\\

tcspc_external_markers

Anonymous (anonymous@undisclosed.example.com) — 2020-09-14T16:01:28+00:00

2019/07/09 13:39		current
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	* PI Controller: E725		* PI Controller: E725
	* PI Controller: E710 \\		* PI Controller: E710 \\
		+	* PI Controller: E727 \\
		+	* Controller: C867.2U2 (PI wide range scanner)
		+	* Controller: C867.262 (PI wide range scanner)
		+
	which have been used in PicoQuant systems before [[http://tcspc.com/doku.php/software:supported_mt200_pc_configuration_for_symphotime_64\|and also MadCity labs can help you getting the proper cable for their stages. ]]		which have been used in PicoQuant systems before [[http://tcspc.com/doku.php/software:supported_mt200_pc_configuration_for_symphotime_64\|and also MadCity labs can help you getting the proper cable for their stages. ]]
		+
		+

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 ======

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 }}

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 =====

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]].
-

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.

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
Line 1:		Line 1:
-		+	{{tag>howto tutorial FLIM analysis SymPhoTime MT LSM_upgrade reconvolution fitting}}
-	{{tag>howto tutorial FLIM analysis SPT SymPhoTime MT LSM_upgrade reconvolution fitting}}	+


Line 11:		Line 10:
	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]].

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;

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 =====

pile-up_effect

Anonymous (anonymous@undisclosed.example.com) — 2015-06-03T15:09:55+00:00

2015/03/26 12:10		current
Line 1:		Line 1:
-	{{tag>pile-up TCSPC}}	+	{{tag> TCSPC pile-up dead_time}}
		+	~~TOC~~

	====== Pile-Up Effect ======		====== Pile-Up Effect ======
Line 30:		Line 30:

	{{ :glossary:pile-up_example.png?600 \|}}		{{ :glossary:pile-up_example.png?600 \|}}
-

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 =====

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 =====
Line 43:		Line 43:
	- 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.

dead_time

Anonymous (anonymous@undisclosed.example.com) — 2015-06-03T15:04:41+00:00

2026/04/11 16:48	current
Line 1:	Line 1:
	+	====== Dead Time ======
	+	In TCSPC the term dead time refers to the time the TCSPC system needs armed again after detecting an event. During the dead time the TCSPC system is blind.

	+	If for example two photons are detected with the dead time of the TCSPC device the second photon will be lost. This leads to the so called [[glossary:Pile-up Effect]].
	+
	+	Note that not only TCSPC devices but also photon counting devices exhibit dead times.

pre-histogrammed_image

Anonymous (anonymous@undisclosed.example.com) — 2023-11-15T14:13:50+00:00

2016/03/03 09:58		current
Line 1:		Line 1:
	====== Structure of the pre-histogrammed Image Data File======		====== Structure of the pre-histogrammed Image Data File======
-	****	+
	^Data Item ^^^^Type ^Description ^		^Data Item ^^^^Type ^Description ^
	\|PixX \|\|\|\|''int32'' \|pixels in X-direction \|		\|PixX \|\|\|\|''int32'' \|pixels in X-direction \|
Line 16:		Line 16:


-	For an example in python see here: https://github.com/PicoQuant/pre-histogrammed-Image-Data-File	+	For an example in Python and Matlab see here: https://github.com/PicoQuant/pre-histogrammed-Image-Data-File

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
Line 81:		Line 81:

	* 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".

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 ======

configuring_symphotime64_after_installation

Anonymous (anonymous@undisclosed.example.com) — 2023-02-17T12:39:29+00:00

2015/01/22 14:37		current
Line 1:		Line 1:
-		+	{{tag> SymPhoTime software howto}}
-	{{tag> SPT64 SymPhoTime software howto}}	+
	~~TOC~~		~~TOC~~
	====== Configuring SymPhoTime64 ======		====== Configuring SymPhoTime64 ======
Line 44:		Line 43:
	* Def.: Default value. This value will be selected when the laser is selected via the SymPhoTIme64 as a statrting value for the intensity of the driver.		* Def.: Default value. This value will be selected when the laser is selected via the SymPhoTIme64 as a statrting value for the intensity of the driver.
	* CW: enable if the laser can run in CW mode		* CW: enable if the laser can run in CW mode
-	* Max. frequency: Maximum allowed repetition rate for the laer in the pulsed mode	+	* Max. frequency: Maximum allowed repetition rate for the laser in the pulsed mode
	<WRAP center tip 40%>		<WRAP center tip 40%>
	Do not forget to press "Save Head" after setting up each Laser Head"		Do not forget to press "Save Head" after setting up each Laser Head"

hydraharp_400

Anonymous (anonymous@undisclosed.example.com) — 2015-09-23T11:43:51+00:00

2015/09/23 11:43		current
Line 3:		Line 3:
	The HydraHarp 400 is a high-end, easy to use, plug and play Time Interval Analyzer (TIA) and Time-Correlated Single Photon Counting (TCSPC) system with scalability for multiple channels. It is connected to a PC through a USB 3.0 high speed interface.		The HydraHarp 400 is a high-end, easy to use, plug and play Time Interval Analyzer (TIA) and Time-Correlated Single Photon Counting (TCSPC) system with scalability for multiple channels. It is connected to a PC through a USB 3.0 high speed interface.

-	See [[http://www.picoquant.com/products/category/tcspc-and-time-tagging-modules/hydraharp-400-multichannel-picosecond-event-timer-tcspc-module\|here]] for more information	+	See the [[http://www.picoquant.com/products/category/tcspc-and-time-tagging-modules/hydraharp-400-multichannel-picosecond-event-timer-tcspc-module\|picoquant.com website]] for more information.

	===== Software =====		===== Software =====
-	Like all PicoQuant TCSPC devices the PicoHarp 300 comes with dedicated software for acquisition of [[glossary:T3-mode]] and [[glossary:T2-mode]] data.	+	Like all PicoQuant TCSPC devices the HydraHarp 400 comes with dedicated software for acquisition of [[glossary:T3-mode]] and [[glossary:T2-mode]] data.

	A library for custom written software as well as systems software for FLIM and FCS is available as an option. A Compilation of different software options can be found [[:Software\|here]].		A library for custom written software as well as systems software for FLIM and FCS is available as an option. A Compilation of different software options can be found [[:Software\|here]].

picoharp_300

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:55:34+00:00

2015/02/10 16:52		current
Line 4:		Line 4:

	See [[http://www.picoquant.com/products/category/tcspc-and-time-tagging-modules/picoharp-300-stand-alone-tcspc-module-with-usb-interface#product-details=description\| here ]] for technical data, spec sheets etc		See [[http://www.picoquant.com/products/category/tcspc-and-time-tagging-modules/picoharp-300-stand-alone-tcspc-module-with-usb-interface#product-details=description\| here ]] for technical data, spec sheets etc
		+
		+
	===== Software =====		===== Software =====
-	see [[:software]]	+	Like all PicoQuant TCSPC devices the PicoHarp 300 comes with dedicated software for acquisition of [[glossary:T3-mode]] and [[glossary:T2-mode]] data.
		+
		+	A library for custom written software as well as systems software for FLIM and FCS is available as an option. A Compilation of different software options can be found [[:Software\|here]].

some_origins_of_multiexponetial_decays_for_single_dyes

Anonymous (anonymous@undisclosed.example.com) — 2019-03-19T12:31:43+00:00

start

Anonymous (anonymous@undisclosed.example.com) — 2026-03-23T07:40:50+00:00

2021/06/17 14:47		current
Line 48:		Line 48:
	===== Data Acquisition and Analysis Software=====		===== Data Acquisition and Analysis Software=====

-	[[Software#Commercial \| PicoQuant SymPhoTime Software (SPT32/SPT64)]]	+	[[https://helpdesk.picoquant.com/help/en-us/30-faq/65-open-source-software-for-picoquant-data-analysis-and-acquisition\|Open-Source Software for PicoQuant Data Analysis and Acquisition]]
-		+
-	[[Software#Commercial \| Phasor Analysis]]	+
-		+
-	[[Software#Open Source Software]]	+
	===== Fluorophores and Samples =====		===== Fluorophores and Samples =====

how_can_i_analyze_my_data_using_non-picoquant_software

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T15:49:47+00:00

2025/01/31 09:01		current
Line 1:		Line 1:
	====== How can I analyze my data using non-PicoQuant software? ======		====== How can I analyze my data using non-PicoQuant software? ======

-	{{tag> file_format ptu open_source faq}}	+	{{tag> faq file_format ptu open_source analysis software}}

	The PicoQuant raw-data format is freely accessible. PicoQuant has published some demo scripts for reading the different TCSPC/TTTR data formats on Github:		The PicoQuant raw-data format is freely accessible. PicoQuant has published some demo scripts for reading the different TCSPC/TTTR data formats on Github:

differential_count_rate

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

2017/09/23 22:43		current
Line 15:		Line 15:
	A frequently asked question is: "//Why we have to consider some differential count rate if we are detecting rarely one photon (count) per excitation cycle, anyway? Detection probability of two or more photons in one cycle is completely negligible!//"		A frequently asked question is: "//Why we have to consider some differential count rate if we are detecting rarely one photon (count) per excitation cycle, anyway? Detection probability of two or more photons in one cycle is completely negligible!//"

-	Not really. In case of pulsed signals the average count rate is a misleading quantity. An average count rate value does not take into account when and how those photons are emitted and detected. Interpreting a 100 kcps intensity as a constant emission rate (Poisson mean rate, in maths terms) is a misconception. The physics of the measurement is completely different. These photons are obviously not emitted evenly, one by one over the whole one second period. They arrive to the detector bunched, as flashes. These are short time intervals with huge photon density (rate), separated by long "dark", quiet periods. Getting a count at "1% of SYNC rate" merely means a sampling rate of a signal with very inhomogeneous time distribution.	+	Not really. In case of pulsed signals the average count rate is a misleading quantity. An average count rate value does not take into account when and how those photons are emitted and detected. Interpreting a 100 kcps intensity as a constant emission rate (Poisson mean rate, in math terms) is a misconception. The physics of the measurement is completely different. These photons are obviously not emitted evenly, one by one over the whole one second period. They arrive to the detector bunched, as flashes. These are short time intervals with huge photon density (rate), separated by long "dark", quiet periods.

-	In mathematical terms, "average count rate of 1..2% of SYNC rate" means the overall detection probability, integrated over the whole duration of a measurement. The concept of //differential count rate// is related to the //probability density function//.	+	In yet another words, achieving a final count rate at "1% of SYNC rate" is a result of sparse sampling with dead time. The sampled signal features much higher photon density, but lasts only for a short time.
		+
		+	In mathematical terms, "average count rate of 1..2% of SYNC rate" means the overall detection probability, integrated over the whole duration of a measurement. The concept of //differential count rate// is related to the //probability density function//. The detected signal in TCSPC has a very inhomogeneous time distribution.

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

tttr

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:42:50+00:00

2015/02/10 16:41		current
Line 6:		Line 6:
	The [[Products:TimeHarp 200]] (also the [[Products:TimeHarp 100]]), the [[Products:PicoHarp 300]] [[Products:HydraHarp 400]] support TTTR mode measurements. With the the [[Products:PicoHarp 300]] a slight shift in the nomenclature was introduced. Due to its capability of simultaneously recording both signal inputs (in interactive mode used as 'Start' and 'Stop') as two separate TTTR traces with a time resolution of 4ps, a new TTTR mode was introduced, the so called T2-mode. The traditional recording as with the [[Products:TimeHarp 200]] / [[Products:TimeHarp 100]] is called [[T3-mode]]. In [[T2-mode]] both signal inputs are functionally identical.		The [[Products:TimeHarp 200]] (also the [[Products:TimeHarp 100]]), the [[Products:PicoHarp 300]] [[Products:HydraHarp 400]] support TTTR mode measurements. With the the [[Products:PicoHarp 300]] a slight shift in the nomenclature was introduced. Due to its capability of simultaneously recording both signal inputs (in interactive mode used as 'Start' and 'Stop') as two separate TTTR traces with a time resolution of 4ps, a new TTTR mode was introduced, the so called T2-mode. The traditional recording as with the [[Products:TimeHarp 200]] / [[Products:TimeHarp 100]] is called [[T3-mode]]. In [[T2-mode]] both signal inputs are functionally identical.

-	Today recording of T3 and T2 mode files is possible with all recent PicoQuant TCSPC devices ([[Products:TimeHarp 260]], [[Products:PicoHarp 300]] and [[Products:HydraHarp 400]] and their included software, analysis is supported mainly by the [[software:SymPhoTime]] software package.	+	Today recording of [[glossary:T3-mode]] and [[glossary:T2-mode]] files is possible with all recent PicoQuant TCSPC devices ([[Products:TimeHarp 260]], [[Products:PicoHarp 300]] and [[Products:HydraHarp 400]] and their included software, analysis is supported mainly by the [[software:SymPhoTime]] software package.

	Due to its capability of including external TTL pulses as markers in the stream of photon events TTTR mode is suitable for [[FLIM]] measurements. The markers are used to synchronise the photon events with a scanning process allowing to sort the photons into image pixels.		Due to its capability of including external TTL pulses as markers in the stream of photon events TTTR mode is suitable for [[FLIM]] measurements. The markers are used to synchronise the photon events with a scanning process allowing to sort the photons into image pixels.

poisson_distribution

Anonymous (anonymous@undisclosed.example.com) — 2014-04-09T20:47:01+00:00

2014/01/21 16:48		current
Line 6:		Line 6:
	$$P_{\nu}(n)={{\nu^n~e^{-\nu}}\over{n!}}$$		$$P_{\nu}(n)={{\nu^n~e^{-\nu}}\over{n!}}$$

-	The Poisson distribution is of interest especially for [[TCSPC]]: The expected number of photons in any TCSPC channel is given by the 'real' decay (including convolution with the IRF etc.), while the stochastic nature of the measurement process (either a photon is detected or it is not) introduces noise, which follows a Poisson distribution. In the limit of large ${\nu}^{}_{}$ the Poisson distribution approaches a [[Gaussian distribution]] with a width of $\sqrt{\nu}$ centred around ${\nu}^{}_{}$.	+	The Poisson distribution is of interest especially for [[TCSPC]]: The expected number of photons in any TCSPC channel is given by the 'real' decay (including convolution with the IRF etc.), while the stochastic nature of the measurement process (either a photon is detected or it is not) introduces noise, which follows a Poisson distribution. In the limit of large ${\nu}^{}_{}$ the Poisson distribution approaches a [[wp>Normal_distribution\|Gaussian distribution]] with a width of $\sqrt{\nu}$ centred around ${\nu}^{}_{}$.

-	In the Gaussian limit [[least squares]] [[fitting]] may be applied, otherwise [[MLE]] [[fitting]] is preferable.	+	In the Gaussian limit [[least squares]] [[wp>Regression_analysis\|fitting]] may be applied, otherwise [[MLE]] [[wp>Regression_analysis\|fitting]] is preferable.

tcspc

Anonymous (anonymous@undisclosed.example.com) — 2014-01-28T13:06:31+00:00

2013/08/09 15:11		current
Line 1:		Line 1:
	====== TCSPC ======		====== TCSPC ======

-	TCSPC stands for Time Correlated Single Photon Counting: A stream of photons is recorded, each photon with respect to a (often periodical) signal, e.g. a repeated laser pulse. The single photon events are sorted into histogram channels that correspond to the time that elapsed since the last excitation pulse. It is a time domain method of investigating time resolved fluorescence. [[Products:TimeHarp 260]], [[Products:HydraHarp 400]] and [[Products:PicoHarp 300]] use TCSPC.	+	TCSPC stands for Time Correlated Single Photon Counting: A stream of photons is recorded, each photon with respect to a (often periodical) signal, e.g. a repeated laser pulse. The single photon events are sorted into histogram channels that correspond to the time that elapsed since the last excitation pulse. It is a time domain method for investigating time resolved fluorescence.

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]]

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.
-

supported_mt200_pc_configuration_for_symphotime_64

Anonymous (anonymous@undisclosed.example.com) — 2023-02-17T12:44:38+00:00

2023/02/17 12:40		current
Line 1:		Line 1:
-	{{tag>SymPhoTime MT200 MicroTime Scanner PI Physik_Instrumente E725 E710 MadCityLabs Mad_City_Labs PC}}	+	{{tag>SymPhoTime MicroTime200 Scanner PI Physik_Instrumente E725 E710 MadCityLabs Mad_City_Labs PC}}
	~~TOC~~		~~TOC~~

privacy_policy

Anonymous (anonymous@undisclosed.example.com) — 2019-12-03T15:49:17+00:00

2019/12/03 13:01		current
Line 198:		Line 198:

	Grundsätzlich besteht für die betroffene Person die Möglichkeit, einer Erfassung der durch den Matomo erzeugten, auf eine Nutzung dieser Internetseite bezogenen Daten zu widersprechen und eine solche zu verhindern. Hierzu muss die betroffene Person in Ihrem Browser "Do Not Track" einstellen.		Grundsätzlich besteht für die betroffene Person die Möglichkeit, einer Erfassung der durch den Matomo erzeugten, auf eine Nutzung dieser Internetseite bezogenen Daten zu widersprechen und eine solche zu verhindern. Hierzu muss die betroffene Person in Ihrem Browser "Do Not Track" einstellen.
-
-	Ferner besteht für die betroffene Person die Möglichkeit der anonymisierten Datenerhebung durch das Programm Matomo jederzeit mit Wirkung für die Zukunft zu widersprechen, indem sie auf den untenstehenden Link klicken. In diesem Fall wird ein sog. Opt-Out-Cookie im Browser der betroffenen Person abgelegt, was zur Folge hat, dass Matomo keinerlei Sitzungsdaten mehr erhebt. Wenn betroffene Person ihre Cookies löschen, so hat dies jedoch zur Folge, dass auch das Opt-Out-Cookie gelöscht wird und daher von den betroffene Person erneut aktiviert werden muss.

	Die Logs mit den Daten der betroffene Person werden nach spätestens 6 Monaten gelöscht.		Die Logs mit den Daten der betroffene Person werden nach spätestens 6 Monaten gelöscht.
-	<html><iframe src="https://www.picoquant.com/piwik/index.php?module=CoreAdminHome&action=optOut&language=en" style="border: 0; height: auto; width: 100%;"></iframe></html>
-
-	{{url>https://www.picoquant.com/piwik/index.php?module=CoreAdminHome&action=optOut&language=en}}

	Weitere Informationen und die geltenden Datenschutzbestimmungen von Matomo können unter [[https://matomo.org/privacy/]] abgerufen werden.		Weitere Informationen und die geltenden Datenschutzbestimmungen von Matomo können unter [[https://matomo.org/privacy/]] abgerufen werden.

supported_th260_pc

Anonymous (anonymous@undisclosed.example.com) — 2017-09-20T15:42:57+00:00

2017/04/07 18:03		current
Line 1:		Line 1:
		+	/*
	{{tag> PC TH260 Harps TCSPC}}		{{tag> PC TH260 Harps TCSPC}}
	~~TOC~~		~~TOC~~
Line 37:		Line 38:
	\|HP ENVY 700-056 \|AMD A75 FCH\| \|		\|HP ENVY 700-056 \|AMD A75 FCH\| \|
	\|Dell Precision T3400 \|Intel X38 \| Installed in SLOT 3 , PCIe 8x(4,1)\|		\|Dell Precision T3400 \|Intel X38 \| Installed in SLOT 3 , PCIe 8x(4,1)\|
		+	\|Dell Optiplex 990 \|Intel X38 \| PCIe 8x \| /* See ticket 828080*\

	===== not-working mainboards / computer =====		===== not-working mainboards / computer =====
Line 42:		Line 44:
	^ Computer ^Chipset ^Comment^		^ Computer ^Chipset ^Comment^
	\|DELL Vostro 630 \| \| \|		\|DELL Vostro 630 \| \| \|
		+	\|DELL Optiplex 9010 \| \| \|
	\|DELL Optiplex 9020 Model Minitower \| \| \|		\|DELL Optiplex 9020 Model Minitower \| \| \|
	\|DELL Optiplex 7020 \| \| \|		\|DELL Optiplex 7020 \| \| \|
Line 47:		Line 50:
	\|HP 200-G1 (Model L5L54US#ABA) \| \| \|		\|HP 200-G1 (Model L5L54US#ABA) \| \| \|
	\|Lenovo ThinkServer TS140 \| \| \|		\|Lenovo ThinkServer TS140 \| \| \|
		+
		+	*/

synchrotron_applications

Anonymous (anonymous@undisclosed.example.com) — 2017-05-17T08:31:53+00:00

2014/07/09 17:27		current
Line 2:		Line 2:
	====== Synchrotron Applications ======		====== Synchrotron Applications ======

-	TCSPC devices (such as [[products:PicoHarp300]]) are also used to measure the temporal distribution of sychrotron radiation that corresponds to the temporal distribution of the electron beam that generate it and thus the filling pattern. We measure the temporal distribution of the radiation by measuring the arrival time of a photon to the laboratory with respect to a trigger given by the machine timing. So in this optics we are certainly measuring the time of flight of photons.	+	TCSPC devices (such as [[products:PicoHarp 300]]) are also used to measure the temporal distribution of sychrotron radiation that corresponds to the temporal distribution of the electron beam that generate it and thus the filling pattern. We measure the temporal distribution of the radiation by measuring the arrival time of a photon to the laboratory with respect to a trigger given by the machine timing. So in this optics we are certainly measuring the time of flight of photons.

-	Software to integrate the [[products:PicoHarp300]] into the [[http://www.tango-controls.org/\|Tango Control Software]] used at the [[http://www.albasynchrotron.es/en\|ALBA Synchrotron]] is being developed.	+	Software to integrate the [[products:PicoHarp 300]] into the [[http://www.tango-controls.org/\|Tango Control Software]] used at the [[http://www.albasynchrotron.es/en\|ALBA Synchrotron]] is being developed.

	The [[http://sourceforge.net/p/tango-ds/code/HEAD/tree/DeviceClasses/MeasureInstruments/PicoHarp300/\|development]] has been in split into smaller parts:		The [[http://sourceforge.net/p/tango-ds/code/HEAD/tree/DeviceClasses/MeasureInstruments/PicoHarp300/\|development]] has been in split into smaller parts:
	* A cython library to access the original's manufacturer library from python and in a pythonic way,		* A cython library to access the original's manufacturer library from python and in a pythonic way,
	* An agent in the [[https://www.tango-controls.org/\|Tango distributed control system]] that manage the instrument,		* An agent in the [[https://www.tango-controls.org/\|Tango distributed control system]] that manage the instrument,
-	* A [[http://taurus-scada.org/\|Taurus gui]] with access to the instrument from the control system	+	* A [[http://taurus-scada.org/\|Taurus GUI]] with access to the instrument from the control system

	There is a development now for another agent in the DCS to process the histogram given by the PicoHarp300 instrument to have the distribution mention before.		There is a development now for another agent in the DCS to process the histogram given by the PicoHarp300 instrument to have the distribution mention before.

	Please check [[http://www.picoquant.com/applications/category/metrology/bunch-purity-measurements]] for other Synchrotron-related applications.		Please check [[http://www.picoquant.com/applications/category/metrology/bunch-purity-measurements]] for other Synchrotron-related applications.

contributing

Anonymous (anonymous@undisclosed.example.com) — 2018-02-19T08:06:13+00:00

2014/04/11 07:32		current
Line 11:		Line 11:
	Links that don't have an article yet will be shown in red. This makes it easy for others to just start this article.		Links that don't have an article yet will be shown in red. This makes it easy for others to just start this article.

		+	====== Most searched words ======
		+	~~SEARCHCLOUD~~

	====== Improve an article ======		====== Improve an article ======

novaflim_calculated_irf

Anonymous (anonymous@undisclosed.example.com) — 2025-03-11T08:19:29+00:00

2025/02/18 13:31		current
Line 1:		Line 1:
	====== NovaFLIM Calculated IRF ======		====== NovaFLIM Calculated IRF ======
		+	{{tag> faq software novaflim IRF}}
		+

	In the Nova Flim the IRF is parameterized as a combination of two Gaussian curves (a total of 5 fit parameters). These parameters are adjusted during Initial Fit.		In the Nova Flim the IRF is parameterized as a combination of two Gaussian curves (a total of 5 fit parameters). These parameters are adjusted during Initial Fit.
Line 22:		Line 24:
	* Height of Gaussian curve 2 (limited to the height of Gaussian curve 1)		* Height of Gaussian curve 2 (limited to the height of Gaussian curve 1)

-	This model is the result of extensive internal development, testing, and refinement. By optimizing these parameters, the IRF model can accurately represent the measured response of the system.	+	The IRF is determined only during the initial TCSPC fit, which means it may vary depending on the different exponential components. During the pixelwise fitting in FLIM, this IRF is applied without any modifications. Consequently, all image points are fitted using the same fixed IRF established during the initial fit.
		+
		+	This model is the result of extensive internal development, testing, and refinement. By optimizing these parameters, the IRF model can accurately represent the measured response of the system.

start

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T09:43:52+00:00

2025/02/14 09:36		current
Line 8:		Line 8:
	==== Why do I not get the same count rates on both SPAD detectors? ====		==== Why do I not get the same count rates on both SPAD detectors? ====

-	In this article we assume that both detectors are SPADs of the same kind (both Excelitas SPADs, or both PDM-SPADs, or both tau-SPADs).	+	In this article we assume that both detectors are SPADs of the same kind (both Excelitas SPADs, or both PDM-SPADs, or both tau-SPADs).

-	If you are using either a proper (see below) 50/50 cube beamsplitter to split the signal between two detectors or a 100% mirror to direct the signal to one detector and then the other, but still observe very different count rates on the two detectors, this discrepancy could be due to the following reasons:	+	If you are using either a proper (see below) 50/50 beamsplitter to split the signal between two detectors or a 100% mirror to direct the signal to one detector and then the other, but still observe very different count rates on the two detectors, this discrepancy could be due to the following reasons:

	1. Misalignment of the Lens in Front of the SPAD:		1. Misalignment of the Lens in Front of the SPAD:
Line 33:		Line 33:

	- Use a concentrated dye solution and ensure the same emission filter is mounted in front of each detector, or place the emission filter before the splitter (holder before the pinhole).		- Use a concentrated dye solution and ensure the same emission filter is mounted in front of each detector, or place the emission filter before the splitter (holder before the pinhole).
-	- Direct all light to Detector 1 and record the count rate (the count rate should be between 100 kcps and 700 kcps - adjust the laser power accordingly).	+	- Direct all light to Detector 1 and record the count rate (the count rate should be between 100 kcps and 700 kcps - adjust the laser power accordingly).
	- Use a 100% mirror to redirect all light to Detector 2 and record the count rate.		- Use a 100% mirror to redirect all light to Detector 2 and record the count rate.
	- Compare the count rates of Detector 1 and Detector 2. If the detectors are of the same type, similar count rates are expected.		- Compare the count rates of Detector 1 and Detector 2. If the detectors are of the same type, similar count rates are expected.
-	- If the detectors are the same type, and the count rate difference is more than 10%, the alignment on the detector with the lower countrate should be checked.	+	* If the count rate difference is more than 10%, the alignment on the detector with the lower countrate should be checked and maximized.
		+	* Follow the "Everyday Alignment" Procedure from the MicroTime 200 manual to correctly align the lens. It might be that also "Fundamental Alignment" is necessary.
	- Introduce the beamsplitter and measure the combined count rate from both detectors.		- Introduce the beamsplitter and measure the combined count rate from both detectors.
		+	* The sum of the signal on both detectors should be the same as from the previous measurement (i.e. 100% going to Detector 1 or 2).
		+	* If it is not (again, allow for 10% error), check the alignment.

-	If the sum	+	If you have followed these instructions, and the light is not split 50/50, then this is likely a property of the beamsplitter. Make sure that is it the correct kind of beamsplitter (see above), and/or contact us for advice.
-		+
-		+
-	By following these steps, you can determine if the discrepancy in count rates is due to misalignment or the characteristics of the beam-splitter.	+
	===== SymPhoTime =====		===== SymPhoTime =====

fluorescence_correlation_spectroscopy-_a_short_introduction

Anonymous (anonymous@undisclosed.example.com) — 2024-10-11T08:30:57+00:00

2023/02/17 12:39		current
Line 1:		Line 1:
-	{{tag>MT200 MircoTime LSM Theory SymPhoTime FCS Correlation}}	+	{{tag>MicroTime LSM Theory SymPhoTime FCS Correlation}}

-	~~TOC~~

	====== Introduction to Fluorescence Correlation Spectroscopy ======		====== Introduction to Fluorescence Correlation Spectroscopy ======

bifl

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:34:08+00:00

2013/08/06 14:36		current
Line 3:		Line 3:
	BIFL stands for Burst Integrated Fluorescence Lifetime: The photon events of a complete burst are accumulated into a [[TCSPC]] histogram which is used for lifetime analysis methods afterwards. Usually a burst is defined as a time interval for which the intensity of a time trace or [[MCS]] measurement is above a chosen threshold. Evaluation of all bursts present in an [[MCS]] trace, for example, results in a lifetime histogram.		BIFL stands for Burst Integrated Fluorescence Lifetime: The photon events of a complete burst are accumulated into a [[TCSPC]] histogram which is used for lifetime analysis methods afterwards. Usually a burst is defined as a time interval for which the intensity of a time trace or [[MCS]] measurement is above a chosen threshold. Evaluation of all bursts present in an [[MCS]] trace, for example, results in a lifetime histogram.

-	BIFL is supported by the [[SymPhoTime]] software, alongside with a variety of burst related techniques.	+	BIFL is supported by the [[software:SymPhoTime]] software, alongside with a variety of burst related techniques.

fwhm

Anonymous (anonymous@undisclosed.example.com) — 2013-08-06T14:45:49+00:00

2013/08/06 14:45		current
Line 3:		Line 3:
	FWHM stands for Full Width at Half-Maximum: a measure for the broadness of a peak, for example, of the [[IRF]] in an [[TCSPC]] measurement or the focal width(s) in a [[FLIM]] measurement.		FWHM stands for Full Width at Half-Maximum: a measure for the broadness of a peak, for example, of the [[IRF]] in an [[TCSPC]] measurement or the focal width(s) in a [[FLIM]] measurement.

-	see also [[wp FWHM]]	+	see also [[wp>FWHM]]

least_squares

Anonymous (anonymous@undisclosed.example.com) — 2014-04-09T20:43:37+00:00

2014/01/21 16:07		current
Line 2:		Line 2:
	====== Least squares ======		====== Least squares ======

-	Least squares is an optimization paradigm for matching data ('[[fitting]]') with a parametrised model equation. A famous example is the linear regression used for finding the linear equation that best matches a given set of data points.	+	Least squares is an optimization paradigm for matching data ('{{wiki>Regression_analysis\|fitting}}') with a parametrised model equation. A famous example is the linear regression used for finding the linear equation that best matches a given set of data points.

	The least squares measure for the goodness-of-fit is		The least squares measure for the goodness-of-fit is

mcs

Anonymous (anonymous@undisclosed.example.com) — 2014-01-21T16:39:04+00:00

2013/08/09 07:34		current
Line 1:		Line 1:
	====== MCS ======		====== MCS ======

-	MCS stands for Multi Channel Scaler. At PQ this term is used in the following context: Single photon events, as originating from a [[TTTR]] measurement are sorted by means of their macroscopic time into equally spaced time bins. What immediately results is a time trace of the fluorescence intensity. In a subsequent analysis the binned ensemble of photons can be used to form [[TCSPC]] histograms, which in turn can be used for lifetime analysis, time-gated analysis etc.	+	MCS stands for Multi Channel Scaler. At PicoQuant this term is used in the following context: Single photon events, as originating from a [[TTTR]] measurement are sorted by means of their macroscopic time into equally spaced time bins. What immediately results is a time trace of the fluorescence intensity. In a subsequent analysis the binned ensemble of photons can be used to form [[TCSPC]] histograms, which in turn can be used for lifetime analysis, time-gated analysis etc.

-	At PQ, MCS is solely supported by our [[SymPhoTime]] package, at the moment.

residuals

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:35:23+00:00

2014/04/09 20:39		current
Line 1:		Line 1:
	====== Residuals ======		====== Residuals ======

-	In the context of [[least squares]] (or any other [[fitting]] method, e.g. [[MLE]]) the residuals are the difference between a model function and the experimental data. In [[least squares]] usually the weighted residuals trace is shown:	+	In the context of [[least squares]] (or any other [[wp>Regression_analysis\|fitting]] method, e.g. [[MLE]]) the residuals are the difference between a model function and the experimental data. In [[least squares]] usually the weighted residuals trace is shown:

	$$R_{wgt}(t_i)={{D\left(\mbox{model parameters},t_i\right)-D_i^{exp}} \over {w_i}}$$		$$R_{wgt}(t_i)={{D\left(\mbox{model parameters},t_i\right)-D_i^{exp}} \over {w_i}}$$
Line 13:		Line 13:
	$$w_i=\sqrt{D_i^{exp}}$$		$$w_i=\sqrt{D_i^{exp}}$$

-	The resudials trace is of importance within any framework concerned with [[fitting]], as the [[products:SymPhoTime]] software or [[FluoFit]].	+	The resudials trace is of importance within any framework concerned with fitting, as the [[software:SymPhoTime]] software or [[software:FluoFit]].

2ffcs

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

2013/12/03 11:42		current
Line 1:		Line 1:
-	====== 2FFCS ======	+	{{tag> 2fFCS FCS}}
		+	~~TOC~~
		+	====== 2fFCS ======


Line 5:		Line 7:

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

	===== Experimental Setup =====		===== Experimental Setup =====
Line 38:		Line 42:
	Occasionally, pinhole size can be used to adjust amount of photons to change the signal intensity and increase SNR. In addition to the "optimal" 1 AU, Pinhole 1-3 AU is the range of choice. Bigger pinhole give you stronger signal but with the compromised confocal effects.		Occasionally, pinhole size can be used to adjust amount of photons to change the signal intensity and increase SNR. In addition to the "optimal" 1 AU, Pinhole 1-3 AU is the range of choice. Bigger pinhole give you stronger signal but with the compromised confocal effects.

-	PQ usually uses a 50 micron pinhole (1.4 AU) to get maximum detection efficiency, somewhat sacrificing background rejection.	+	PicoQuant usually uses a 50 micron pinhole (1.4 AU) to get maximum detection efficiency, somewhat sacrificing background rejection.

	=== Two Foci ===		=== Two Foci ===

antibunching_measurements

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

2023/09/07 11:54		current
Line 5:		Line 5:
	====== How to Perform Antibunching Measurements ======		====== How to Perform Antibunching Measurements ======

		+	Antibunching Measurements are (normally) measured in T2 mode. In T2 mode, all input channels of the TCSPC electronics are treated the same, also the "Sync" channel. Therefore the sync has to be disabled either by disconneting the cable, or by changing the CFD level or trigger level to such a value that the sync is not recognized any more. i.e. Sync rate = 0 . Directly in the Sepia software, the sync out can also be disabled.
		+
		+	The descriptions below only mention physically disconnecting the sync. This is strictly necessary only for the PicoHarp 300 because it only has two channels.
	===== Emission Filters =====		===== Emission Filters =====

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 =====
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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.

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 ====

select_the_correct_pinhole_size

Anonymous (anonymous@undisclosed.example.com) — 2015-09-11T15:24:14+00:00

2015/09/11 15:20		current
Line 6:		Line 6:
	====== How-to select the correct pinhole size ======		====== How-to select the correct pinhole size ======

-	The pinhole size should be slightly bigger than the image of the Airy disc at the position of the pinhole:	+	The pinhole size should be slightly bigger than the image of the [[wp>Airy Disc]] at the position of the pinhole:

	$$d_{Airy} = (1.22 * lambda / N.A.) * M$$		$$d_{Airy} = (1.22 * lambda / N.A.) * M$$

start

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

2024/12/19 15:56		current
Line 8:		Line 8:
	{{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 =====

microtime

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:24:05+00:00

2015/11/03 14:22		current
Line 18:		Line 18:
	* Complete confocal system with laser combining unit, inverted microscope body and multichannel detection unit		* Complete confocal system with laser combining unit, inverted microscope body and multichannel detection unit
	* Turn-key diode lasers for multicolor excitation from 375 nm to 900 nm		* Turn-key diode lasers for multicolor excitation from 375 nm to 900 nm
-	* Up to 4 truly parallel detection channels using application-optimized detection with [[glossray:SPAD]]s, [[glossary:PMT]]s or [[glossary:Hybrid PMT]]s	+	* Up to 4 truly parallel detection channels using application-optimized detection with [[glossary:SPAD]]s, [[glossary:PMT]]s or [[glossary:Hybrid PMT]]s
-	* Time-Correlated-Single Photon Counting (TCSPC) and TTTR mode for investigation of fast dynamics in FCS and FLIM	+	* Time-Correlated-Single Photon Counting ([[glossary:TCSPC]]) and [[glossary:TTTR]] mode for investigation of fast dynamics in [[glossary:FCS]] and [[glossary:FLIM]]
	* Piezo scanning for 2D- and 3D-lifetime imaging and accurate point positioning		* Piezo scanning for 2D- and 3D-lifetime imaging and accurate point positioning
	* Two optional exit ports for additional hardware, e.g. spectrographs		* Two optional exit ports for additional hardware, e.g. spectrographs

tcspc_electronics

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:49:36+00:00

2026/04/11 16:48	current
Line 1:	Line 1:
	+	====== Electronics for Time Correlated Single Counting ======
	+	for a definition of TCSCP see here: [[glossary:TCSPC]]
	+	===== PicoQuant =====
	+	* [[products:TimeHarp 260]]
	+	* [[products:PicoHarp 300]]
	+	* [[products:HydraHarp 400]]
	+	==== discontinued ====
	+	* [[products:TimeHarp 100]]
	+	* [[products:TimeHarp 2000]]

	+

supported_mt200_pc_configuration_for_symphotime_32

Anonymous (anonymous@undisclosed.example.com) — 2016-08-25T11:24:41+00:00

2015/10/28 09:36		current
Line 1:		Line 1:
-	{{tag> SymPhoTime MT200 MicroTime SPT32}}	+	{{tag> SymPhoTime MT200 MicroTime SPT32 PC}}
	~~TOC~~		~~TOC~~

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	===== Windows 7 (64bit) =====		===== Windows 7 (64bit) =====
-	* not data available yet	+	* not supported for controlling the MicroTime200
	not supported:		not supported:
	* DataTranslation PCI framegrabber - no driver available for 64 bit systems - cannot work neither within nor outside SymPhoTime64 software		* DataTranslation PCI framegrabber - no driver available for 64 bit systems - cannot work neither within nor outside SymPhoTime64 software
		+	* Supported for the "Analysis Mode"

	===== Windows 7 (32bit) =====		===== Windows 7 (32bit) =====

symphotime

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:32:55+00:00

2026/04/11 16:48	current
Line 1:	Line 1:
	+	====== SymPhoTime ======

	+	The SymPhoTime software package is an integrated solution for data acquisition and analysis using PicoQuant's time-resolved confocal microscope MicroTime 200, LSM upgrade kits or TCSPC electronics.
	+
	+	After several very successful years [[software:SymPhoTime32]] has been replaced by the completely rewritten 64bit version [[software:SymPhoTime64]].

symphotime_32

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:47:15+00:00

2026/04/11 16:48	current
Line 1:	Line 1:
	+	====== SymPhoTime 32 ======
	+	SymPhoTime 32 is the name of the now obsolete software package for data acquisition and analysis using PicoQuant's time-resolved confocal microscope [[products:MicroTime 200]], [[products:LSM upgrade kits]] or [[products:TCSPC electronics]].

	+	SymPhoTime has been replace by the 64bit version called [[software:SymPhoTime64]]

symphotime64

Anonymous (anonymous@undisclosed.example.com) — 2015-01-22T14:39:59+00:00

2014/05/23 07:33		current
Line 5:		Line 5:


-	The [[http://www.picoquant.com\|PicoQuant]] SymPhoTime 64 software package is an integrated solution for data acquisition and analysis using PicoQuant's time-resolved confocal microscope MicroTime 200, LSM upgrade kits or [[glossary:TCSPC]] electronics. Its clearly structured layout and powerful analysis routines allows the user to focus on the results rather than on the data processing. The software is designed for a 64 bit operation system and features a graphical user interface (GUI), which guides the user through all necessary steps for an individual analysis or measurement process. Data dependencies are directly visible in the underlying workspace concept. An integrated scripting language ("STUPSLANG") even puts the user in a position to freely add new analysis procedures or customize existing ones.	+	The [[http://www.picoquant.com\|PicoQuant]] SymPhoTime 64 software package is an integrated solution for data acquisition and analysis using PicoQuant's time-resolved confocal microscope [[products:MicroTime]], LSM upgrade kits or [[glossary:TCSPC]] electronics. Its clearly structured layout and powerful analysis routines allows the user to focus on the results rather than on the data processing. The software is designed for a 64 bit operation system and features a graphical user interface (GUI), which guides the user through all necessary steps for an individual analysis or measurement process. Data dependencies are directly visible in the underlying workspace concept. An integrated scripting language ("STUPSLANG") even puts the user in a position to freely add new analysis procedures or customize existing ones.

	For more information, latest version and download link see [[http://www.picoquant.com/products/category/software/symphotime-64-fluorescence-lifetime-imaging-and-correlation-software\|SymPhoTime64]].		For more information, latest version and download link see [[http://www.picoquant.com/products/category/software/symphotime-64-fluorescence-lifetime-imaging-and-correlation-software\|SymPhoTime64]].

2019/03/06 12:44		current
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-	The fluorescence lifetime of a dye measured with a TCSPC spectrometer can be multiexponential due to many reasons. The most obvious cases are due to scattering or presence of impurities. Although less obvious, it is also widely known that in an inhomogeneous media a pure dye will also exhibit a multiexponential decay. Advanced users know that if the decay is not measured with polarizers at magic angle, the rotation correlation time shows up as a second exponential in the decay (the second exponential is in fact the product of the rotation correlation time by the fluorescence lifetime, divided by their sum). And they also know that measuring without polarizers is not equivalent to measure at magic angle... But suspicion may arise when even a pure dye measured at magic angle in a homogeneous media exhibits a multiexponential decay. Is the spectrometer properly adjusted? Are the polarizers properly calibrated? A typical mistake is to measure the [[glossary:IRF]] at the nominal laser wavelength instead of measuring at the ideal wavelength for that specific laser head. Note that all diode lasers heads emit at slightly different wavelengths and each of them have an optimum at which the IRF should be measured. As little as 0.5 nm displacement from their optimum may induce a "non perfect" [[glossary:deconvolution]] fit.	+	The fluorescence lifetime of a dye measured with a TCSPC spectrometer can be multiexponential due to many reasons. The most obvious cases are due to scattering or presence of impurities. Although less obvious, it is also widely known that in an inhomogeneous medium a pure dye will also exhibit a multiexponential decay. Advanced users know that if the decay is not measured with polarizers at magic angle, the rotation correlation time shows up as a second exponential in the decay (the second exponential is in fact the product of the rotation correlation time and the fluorescence lifetime, divided by their sum). And they also know that measuring without polarizers is not equivalent to measuring at magic angle... But suspicion may arise when even a pure dye measured at magic angle in a homogeneous medium exhibits a multiexponential decay. Is the spectrometer properly adjusted? Are the polarizers properly calibrated? A typical mistake is to measure the [[glossary:IRF]] at the nominal laser wavelength instead of measuring at the ideal wavelength for that specific laser head. Note that all diode lasers heads emit at slightly different wavelengths and each of them have an optimum at which the IRF should be measured. As little as 0.5 nm displacement from their optimum may induce a "non perfect" [[glossary:deconvolution]] fit.

	But it is worth noting that even at magic angle in a perfectly aligned spectrometer pure dyes in homogeneous media may exhibit a multiexponential decay. The origin may be physical, like solvent relaxation, or chemical, when the fluorescent molecule undergoes a ground or excited state reaction. In this brief article a few examples are described.		But it is worth noting that even at magic angle in a perfectly aligned spectrometer pure dyes in homogeneous media may exhibit a multiexponential decay. The origin may be physical, like solvent relaxation, or chemical, when the fluorescent molecule undergoes a ground or excited state reaction. In this brief article a few examples are described.
Line 25:		Line 25:


-	The electronic redistribution of electrons due to optical excitation leads in many cases to a different reactivity in the ground and excited states. In other words, a fluorescent molecule which is boring under the dark may become reactive upon excitation. Common excited state reactions are redox (electron transfer) and acid-base (proton transfer) reactions. Depending on the rate of the excited-state rection relative the the original fluorescence lifetime, the observed decay time measured with a TCSPC spectrometer may be single- or multiexponential.	+	The electronic redistribution of electrons due to optical excitation leads in many cases to a different reactivity in the ground and excited states. In other words, a fluorescent molecule which is mostly inert in the dark may become reactive upon excitation. Common excited state reactions are redox (electron transfer) and acid-base (proton transfer) reactions. Depending on the rate of the excited-state reaction relative the the original fluorescence lifetime, the observed decay time measured with a TCSPC spectrometer may be single- or multiexponential.

	Let us consider the reaction in Scheme 2 in different situations:		Let us consider the reaction in Scheme 2 in different situations:
Line 34:		Line 34:
	Scheme 2		Scheme 2

-	//Starting point: The molecule A is promoted to the excited-state where it can react with a molecule X to form the compound B, through a rate constant $k_{AB}$. Once the compound B is formed the back-reaction can occur, with a rate constant $k_{BA}$. Compounds A and B are fluorescent with original fluorescence lifetimes $\tau_A$ and $\tau_B$. Once B decays to the ground state the back-reaction takes place. Hence the system is always in its starting position $(A + X)$ prior to any excitation pulse.//	+	//Starting point: The molecule A is promoted to the excited-state where it can react with a molecule X to form the compound B, through a rate constant $k_{AB}$. Once the compound B is formed, the back-reaction can occur with a rate constant $k_{BA}$. Compounds A and B are fluorescent with original fluorescence lifetimes $\tau_A$ and $\tau_B$. Once B decays to the ground state, the back-reaction takes place. Hence the system is always in its starting position $(A + X)$ prior to any excitation pulse.//

	//Case A) The constant kAB is too slow with respect to $\tau_A$ and $\tau_B$.// In this case the compound A would decay to the ground-sate before the excited-state reaction could take place. The decay measured would be single exponential and coincident with $\tau_A$.		//Case A) The constant kAB is too slow with respect to $\tau_A$ and $\tau_B$.// In this case the compound A would decay to the ground-sate before the excited-state reaction could take place. The decay measured would be single exponential and coincident with $\tau_A$.
Line 60:		Line 60:
	being $Z = \sqrt{(M-Y)^2 + 4 k_{AB} k_{BA}[x]}$		being $Z = \sqrt{(M-Y)^2 + 4 k_{AB} k_{BA}[x]}$

-	$A_1= A_0 [M- (1/\tau_2)] / [(1/\tau_1 – 1/\tau_2]$	+	$A_1= A_0 [M- (1/\tau_2)] / [1/\tau_1 – 1/\tau_2]$

-	$A_2= A_0 [(1/\tau_1)- M] / [(1/\tau_1 – 1/\tau_2]$	+	$A_2= A_0 [(1/\tau_1)- M] / [1/\tau_1 – 1/\tau_2]$

-	$B_1= -A_0 k_{AB}[x] / [(1/\tau_1 – 1/\tau_2]$	+	$B_1= -A_0 k_{AB}[x] / [1/\tau_1 – 1/\tau_2]$

-	$B_2= A_0 k_{AB} [x] / [(1/\tau_1 – 1/\tau_2]$	+	$B_2= A_0 k_{AB} [x] / [1/\tau_1 – 1/\tau_2]$

	being $A_0$ the concentration of $A$ at $t=0$.		being $A_0$ the concentration of $A$ at $t=0$.
Line 72:		Line 72:
	Note from $A(t)$ that, even if $B$ would not be fluorescent, the decay of $A$ will still be biexponential!		Note from $A(t)$ that, even if $B$ would not be fluorescent, the decay of $A$ will still be biexponential!

-	A situation like this may occur with molecules dissolved in an aprotic but hygroscopic media, like acetonirile. Water molecules may diffuse (diffusion happens in the nanosecond time scale) and react with fluorophores bearing proton-transfer groups like -OH or -NH2.	+	A situation like this may occur with molecules dissolved in an aprotic but hygroscopic medium, like acetonitrile. Water molecules may diffuse (diffusion happens in the nanosecond time scale) and react with fluorophores bearing proton-transfer groups like -OH or -NH2.

-	//Case D) Interconversion rate constants kAB and kBA are very quick compared to the intrinsic lifetimes $\tau_A$ and $\tau_B$.// In this case the equations of case C would still apply. But in practice, a quick equilibrium between reactants and product would be established. This means that the concentrations of A and B with respect to each other would always be constant prior to their decay, and hence the whole system could be treated as a single dye. The decay would be single exponential, being an average of $\tau_A$ and $\tau_B$ weighted by their fraction in the equilibrium.	+	//Case D) Interconversion rate constants $k_{AB}$ and $k_{BA}$ are very quick compared to the intrinsic lifetimes $\tau_A$ and $\tau_B$.// In this case the equations of case C would still apply. But in practice, a quick equilibrium between reactants and product would be established. This means that the concentrations of A and B with respect to each other would always be constant prior to their decay, and hence the whole system could be treated as a single dye. The decay would be single exponential, being an average of $\tau_A$ and $\tau_B$ weighted by their fraction in the equilibrium.

-	This situation may happen if compounds A and X were directly in contact prior to excitation, for example throgh ground-state interactions.	+	This situation may happen if compounds A and X were directly in contact prior to excitation, for example through ground-state interactions.
	===== 3) Solvation dynamics =====		===== 3) Solvation dynamics =====


-	Even pure fluorophores in pure solvents may lead to multiexponential decays. This is the case of molecules with strong charge transfer character in polar solvents when undergoing solvation dynamics. In such cases the fluorescence spectra shifts to longer wavelengths in time. Measuring the decay in the blue edge of the steady-state spectrum will lead to a multiexponential decay. The ns lifetimes corresponds to the intrinsic fluorescence lifetime of the fluorophore, whereas the ultrafast components (ps) are related to the rate of the shift. Measuring in the red-flank of the steady-sate spectrum leads to multiexponential decays with positive (intrinsic lifetime, ns) an negative (rate of shift, ps) pre-exponential factors. This is depicted in Scheme 3.	+	Even pure fluorophores in pure solvents may lead to multiexponential decays. This is the case of molecules with strong charge transfer character in polar solvents when undergoing solvation dynamics. In such cases the fluorescence spectrum shifts to longer wavelengths on a picosecond time-scale. Measuring the decay in the blue edge of the steady-state spectrum will lead to a multiexponential decay. The ns lifetime corresponds to the intrinsic fluorescence lifetime of the fluorophore, whereas the ultrafast components (ps) are related to the rate of the shift. Measuring in the red-flank of the steady-sate spectrum leads to multiexponential decays with positive (intrinsic lifetime, ns) and negative (rate of shift, ps) pre-exponential factors. This is depicted in Scheme 3.


	{{:solvation_dynamics.png?800\|}}		{{:solvation_dynamics.png?800\|}}

-	Scheme 3. Left: energetic representation of solvation dynamics. The fluorophore is represented as a sphere with a pointing dipole. Solvent molecules are represented in gray around the fluorophore. Right up : Spectral consequence of solvation dynamics. The fluorescence spectrum shifts in time to lower energies. Right bottom: Decay traces measured in different spectral regions. Blue flanks are multiexponential with positive pre-exponentila factors, red flanks are multiexponential with rising components.	+	Scheme 3. Left : energetic representation of solvation dynamics. The fluorophore is represented as a sphere with a pointing dipole. Solvent molecules are represented in gray around the fluorophore. Right, top : Spectral consequence of solvation dynamics. The fluorescence spectrum shifts in time to lower energies. Right, bottom: Decay traces measured in different spectral regions. Blue curves are multiexponential with positive pre-exponentila factors, red curves are multiexponential with rising components.

-	Before the excitation, the fluorophore is in the ground state S0 , which has a characteristic dipole moment. Solvent molecules, which also have their characteristic dipole moment are oriented in such a way that the interactions dipole-dipole with the fluorophore are as favorable possible. When the fluorophore is prompted to the excited state, its electronic distribution switches almost instantly. At time zero after excitation the solvent molecules remain in their "original" orientation to solvate ground state . The resulting dipole -dipole interactions with the fluorophore are hence less favorable. As a result , the solvent begins to relax to solvate the S1 state and brings the system to a more favorable position. Spectroscopically, this is manifested by the time-shift of the emission spectrum to longer wavelengths. Consider that the Steady-State spectrum is the time integral of all those shifting spectra. Measuring in the blue flank (λ1) will lead to a multiexponential decay: the signal decreases because of the shift and the intrinsic decay. Measuring in the red flank (λ3) will lead to a multiexponential decay with a negative pre-exponential factor: the signal first rises due to the increase in signal due to the displacement, and then decays due to the fluorescence lifetime.	+	Before the excitation, the fluorophore is in the ground state S0 , which has a characteristic dipole moment. Solvent molecules, which also have their characteristic dipole moment are oriented in such a way that the dipole-dipole interactions with the fluorophore are as favorable as possible. When the fluorophore is prompted to the excited state, its electronic distribution switches almost instantly. At time zero after excitation the solvent molecules remain in their "original" orientation. The resulting dipole -dipole interactions with the fluorophore are hence less favorable. As a result, the solvent begins to relax to solvate the S1 state and brings the system to a more favorable position. Spectroscopically, this is manifested by the time-shift of the emission spectrum to longer wavelengths. Consider that the Steady-State spectrum is the time integral of all those shifting spectra. Measuring in the blue flank (λ1) will lead to a multiexponential decay: the signal decreases because of the shift and the intrinsic decay. Measuring in the red flank (λ3) will lead to a multiexponential decay with a negative pre-exponential factor: the signal first rises due to the increase in signal due to the displacement, and then decays due to the fluorescence lifetime.

	In fluid media, solvation dynamics can be described with a multiexponential function spanning from the femtosecond time-scale to tens of picoseconds. Hence, the tail of this process can be monitored with a TCSPC spectrometer equipped with fast detectors such as a [[glossary:MCP]] or a Hybrid-PMT. In viscous media or at low temperatures, the ps tail component slows down to ns, and the process can be monitored with slower detectors, such as standard [[glossary:PMT]].		In fluid media, solvation dynamics can be described with a multiexponential function spanning from the femtosecond time-scale to tens of picoseconds. Hence, the tail of this process can be monitored with a TCSPC spectrometer equipped with fast detectors such as a [[glossary:MCP]] or a Hybrid-PMT. In viscous media or at low temperatures, the ps tail component slows down to ns, and the process can be monitored with slower detectors, such as standard [[glossary:PMT]].