Time-Resolved Fluorescence Wiki

lifetime_fitting_using_the_flim_analysis

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

2014/06/18 14:19		current
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	~~NOTOC~~		~~NOTOC~~

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

flim_fret_calculation_for_multi_exponential_donors

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

2020/05/12 14:44		current
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	~~NOTOC~~		~~NOTOC~~

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

software

Anonymous (anonymous@undisclosed.example.com) — 2025-05-28T07:47:47+00:00

2025/05/28 09:18		current
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	{{tag> software acquisition analysis}}		{{tag> software acquisition analysis}}

-	~~TOC~~

	====== Software ======		====== Software ======

lifetime-fitting_using_the_rapid_reconvolution_algorithm

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

2021/06/12 07:05		current
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-		+	{{tag>howto tutorial FLIM analysis SymPhoTime MT LSM_upgrade reconvolution fitting}}
-	{{tag>howto tutorial FLIM analysis SPT SymPhoTime MT LSM_upgrade reconvolution fitting}}	+

intensity_time_trace_analysis

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

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

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

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

lifetime_fitting_using_the_tcpsc_fitting_script

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

2023/09/08 00:37		current
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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 13:42		current
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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.\\

flim-fret_calculation_for_single_exponential_donors

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

2020/05/06 11:19		current
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	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".

fluorescence_correlation_spectroscopy-_a_short_introduction

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

2023/02/17 13:43		current
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	{{tag>MicroTime LSM Theory SymPhoTime FCS Correlation}}		{{tag>MicroTime LSM Theory SymPhoTime FCS Correlation}}

-	~~TOC~~	+

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

visualizing_dynamics_using_the_multiframe-flim_script

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

2021/10/19 21:59		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 and above) ======		====== Visualizing Dynamics Using the Multi Frame FLIM Analysis (updated for SymPhoTime v2.5 and above) ======

roi_fitting_using_the_flim_script

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

2020/05/13 11:42		current
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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 }}

update

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

2023/02/23 07:24		current
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	===== Introduction =====		===== 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.\\		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.

visualizing_dynamics_with_the_multi_frame_flim_analysis

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

2015/11/03 15:33		current
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	~~NOTOC~~		~~NOTOC~~

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

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

burst_duration_and_fcs_diffusion_time

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

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

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

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

some_origins_of_multiexponetial_decays_for_single_dyes

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

symphotime_tips_and_tricks

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

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

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

create_time_gated_image

Anonymous (anonymous@undisclosed.example.com) — 2020-10-14T12:10:44+00:00

2020/10/14 14:10		current
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-	//{{tag>howto analysis lifetime tutorial SymPhoTime}}//	+	{{tag>howto analysis lifetime tutorial SymPhoTime}}

	~~NOTOC~~		~~NOTOC~~

t3-mode

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:27:57+00:00

2015/02/10 17:27		current
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	- the arrival time of the event pair on the overall experiment time scale (the time tag).		- the arrival time of the event pair on the overall experiment time scale (the time tag).

-	The latter was originally obtained from an independent asynchronous clock. This made it difficult to combine the start-stop timing with the time tag or to know the sync period the event belonged to. For the PicoHarp's T3 Mode a smarter approach was chosen:\\	+	The latter was originally obtained from an independent asynchronous clock. This made it difficult to combine the start-stop timing with the time tag or to know the sync period the event belonged to. For the *Harp's T3 Mode a smarter approach was chosen:\\
	The time tag is now obtained by simply counting sync pulses. From the T3 Mode event records it is therefore possible to precisely determine which sync period a photon event belongs to. Since the sync period is also known precisely, this furthermore allows to reconstruct the arrival time of the photon with respect to the overall experiment time.		The time tag is now obtained by simply counting sync pulses. From the T3 Mode event records it is therefore possible to precisely determine which sync period a photon event belongs to. Since the sync period is also known precisely, this furthermore allows to reconstruct the arrival time of the photon with respect to the overall experiment time.

separation_of_2_species_with

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

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

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

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

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

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

calculate_ratiometric_single_pair_fret_distributions_using_the_pie-fret_script

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

2014/06/18 18:06		current
Line 15:		Line 15:
	Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\		Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\
	The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\		The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\
-	To record such traces, usually single molecule sensitive detectors as [[glossary:spad\|SPAD]] or Hybrid-PMA are necessary to successfully detect single molecule fluorescence.\\	+	To record such traces, usually single molecule sensitive detectors as [[glossary:spad\|SPAD]] or [[glossary: Hybrid PMT]]s are necessary to successfully detect single molecule fluorescence.\\
	For the pulsed interleaved excitation ([[glossary:pie\|PIE]]) analysis of the script, pulsed excitation using two lasers is required, one laser exciting the donor, the other the acceptor dye. Usually, this is achieved using the multichannel laser driver PDL828 “Sepia II” in combination with two pulsed diode lasers.		For the pulsed interleaved excitation ([[glossary:pie\|PIE]]) analysis of the script, pulsed excitation using two lasers is required, one laser exciting the donor, the other the acceptor dye. Usually, this is achieved using the multichannel laser driver PDL828 “Sepia II” in combination with two pulsed diode lasers.

how_to_measure_the_instrument_response_function_irf

Anonymous (anonymous@undisclosed.example.com) — 2023-09-07T22:55:03+00:00

2023/09/08 00:54		current
Line 22:		Line 22:
	===== Using samples with ultrafast decay =====		===== Using samples with ultrafast decay =====

-	Some detectors (particularly SPADs) have wavelength dependent timing response. In this case an IRF recorded at the excitation wavelength may not be useful for precise reconvolution. The solution is to acquire the IRF at the fluorescence wavelength, or at least spectrally closer to the fluorescence emission.	+	Some detectors (particularly MPD SPADs) have a wavelength dependent timing response. In this case an IRF recorded at the excitation wavelength may not be useful for precise reconvolution. The solution is to acquire the IRF at the fluorescence wavelength, or at least spectrally closer to the fluorescence emission wavelength.

	==== General recipe ====		==== General recipe ====

microtime

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

2015/11/03 15:23		current
Line 19:		Line 19:
	* 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 [[glossary: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 ([[glossary:TCSPC]]) and [[glossary: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

avoid_pile_up_effect_in_flim_measurements

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

2015/03/26 13:10		current
Line 41:		Line 41:
	* Determine the number of photons via the data reader (right mouse click) in a very bright pixel within the FastFLIM image		* Determine the number of photons via the data reader (right mouse click) in a very bright pixel within the FastFLIM image
	* Calculate the count rate for this bright pixel: Divide the number of photons per pixel by the exposure time per pixel		* Calculate the count rate for this bright pixel: Divide the number of photons per pixel by the exposure time per pixel
		+
		+	===== Differential Count Rate =====
		+	see [[glossary:Differential Count Rate]]

calculate_ratiometric_single_pair_fret_distributions

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

2014/06/18 17:56		current
Line 15:		Line 15:
	Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\		Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\
	The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\		The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\
-	To record such traces, usually single molecule sensitive detectors as SPAD or Hybrid PMA are necessary to successfully detect single molecule fluorescence.\\	+	To record such traces, usually single molecule sensitive detectors as [[glossary:SPAD]] or [[glossary:Hybrid PMT]]s are necessary to successfully detect single molecule fluorescence.\\

	\\		\\

t2-mode

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:24:41+00:00

2015/02/10 17:22		current
Line 1:		Line 1:
	====== T2-mode ======		====== T2-mode ======

-	A special measurement mode of the [[Products:PicoHarp 300]]	+	A special measurement mode of the [[Products:PicoHarp 300]], [[Products:HydraHarp 400]] and [[Products:TimeHarp 260]]


Line 7:		Line 7:
	In T2 Mode both signal inputs of the PicoHarp 300 are functionally identical. There is no dedication of input channel 0 to a [[SYNC]] signal. Usually both inputs are used to connect photon detectors. The events from both channels are recorded independently and treated equally. In each case an event record is generated that contains information about the channel it came from and the arrival time of the event with respect to the overall measurement start. The timing is recorded with 4 ps resolution. Each T2 Mode event record consists of 32 bits. There are 4 bits for the channel number and 28 bits for the time-tag. Routing is not supported. If the time tag overflows, a special overflow marker record is inserted in the data stream, so that upon processing of the data stream a theoretically infinite time span can be recovered at full resolution. [[Dead time]]s exist only within each channel (95 ns typ.) but not across the channels. Therefore, cross correlations can be calculated down to zero lag time. This allows powerful new application such as [[FCS]] with lag times from picoseconds to hours. Autocorrelations can also be calculated at the full resolution but of course only starting from lag times larger than the [[dead time]].		In T2 Mode both signal inputs of the PicoHarp 300 are functionally identical. There is no dedication of input channel 0 to a [[SYNC]] signal. Usually both inputs are used to connect photon detectors. The events from both channels are recorded independently and treated equally. In each case an event record is generated that contains information about the channel it came from and the arrival time of the event with respect to the overall measurement start. The timing is recorded with 4 ps resolution. Each T2 Mode event record consists of 32 bits. There are 4 bits for the channel number and 28 bits for the time-tag. Routing is not supported. If the time tag overflows, a special overflow marker record is inserted in the data stream, so that upon processing of the data stream a theoretically infinite time span can be recovered at full resolution. [[Dead time]]s exist only within each channel (95 ns typ.) but not across the channels. Therefore, cross correlations can be calculated down to zero lag time. This allows powerful new application such as [[FCS]] with lag times from picoseconds to hours. Autocorrelations can also be calculated at the full resolution but of course only starting from lag times larger than the [[dead time]].

-	The 32 bit event records are queued in a [[FIFO]] (First In First Out) buffer capable of holding up to 256 k event records. The FIFO input is fast enough to accept records at the full speed of the time-to-digital converters (up to 10 Mcps each). This means, even during a fast burst no events will be dropped except those lost in the [[dead time]] anyhow. The FIFO output is continuously read by the host PC, thereby making room for fresh incoming events. Even if the average read rate of the host PC is limited, bursts with much higher rate can be recorded for some time. Only if the average count rate over a long period of time exceeds the readout speed of the PC, a FIFO overrun could occur. In case of a FIFO overrun the measurement must be aborted because data integrity cannot be maintained. However, on a modern and well configured PC with Windows 2000 or XP a sustained average count rate over 4 Mcps is possible. This total transfer rate must be shared by the two input channels. For all practically relevant fluorescence detection applications the effective rate per channel is more than sufficient.	+	The 32 bit event records are queued in a [[FIFO]] (First In First Out) buffer capable of holding up to 256 k event records. The FIFO input is fast enough to accept records at the full speed of the time-to-digital converters (up to 10 Mcps each). This means, even during a fast burst no events will be dropped except those lost in the [[dead time]] anyhow. The FIFO output is continuously read by the host PC, thereby making room for fresh incoming events. Even if the average read rate of the host PC is limited, bursts with much higher rate can be recorded for some time. Only if the average count rate over a long period of time exceeds the readout speed of the PC, a FIFO overrun could occur. In case of a FIFO overrun the measurement must be aborted because data integrity cannot be maintained. However, on a modern and well configured PC a sustained average count rate over 4 Mcps is possible. This total transfer rate must be shared by the two input channels. For all practically relevant fluorescence detection applications the effective rate per channel is more than sufficient.

-	For maximum throughput, T2 Mode data streams are normally written directly to disk. The current PicoHarp software 1.2 does not provide any immediate data visualization during a T2 Mode measurement, except count rate and progress display. However, using custom software it is also possible to analyze incoming data "on the fly". Even on-line correlation can be implemented. Obviously this requires efficient processing and possible restrictions in average count rate. The PicoHarp software installation CD contains demo programs to show how T2 Mode files can be read by custom software. The implementation of custom measurement programs requires the PicoHarp programming library, which is available as a separate option.	+	For maximum throughput, T2 Mode data streams are normally written directly to disk. The current Harp software does not provide any immediate data visualization during a T2 Mode measurement, except count rate and progress display. However, using custom software it is also possible to analyze incoming data "on the fly". Even on-line correlation can be implemented. Obviously this requires efficient processing and possible restrictions in average count rate. The PicoHarp software installation CD contains demo programs to show how T2 Mode files can be read by custom software. The implementation of custom measurement programs requires the Harp programming library, which is available as a separate option.

	Note: From a mathematical point of view, the T2 mode should be able to store 28 bit = 228 = 268435456 time bin data before need to "wrap around". However, the real wrap around time as shown in the demo code is only 210698240. This discrepancy is due to the design of the TDCs. Briefly, the [[TDCs]] include a coarse scaler and an interpolator for the the short times. This interpolator is not working in a binary fashion, which finally leads to the fact that not the full available memory is used. This does not lead to any data loss. To reconstruct the full temporal time trace one only needs to follow the procedures shown in the demo code		Note: From a mathematical point of view, the T2 mode should be able to store 28 bit = 228 = 268435456 time bin data before need to "wrap around". However, the real wrap around time as shown in the demo code is only 210698240. This discrepancy is due to the design of the TDCs. Briefly, the [[TDCs]] include a coarse scaler and an interpolator for the the short times. This interpolator is not working in a binary fashion, which finally leads to the fact that not the full available memory is used. This does not lead to any data loss. To reconstruct the full temporal time trace one only needs to follow the procedures shown in the demo code

	see also [[.:t3-mode]]		see also [[.:t3-mode]]
-

configuring_symphotime64_after_installation

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

2015/01/22 16:45		current
Line 1:		Line 1:
-		+	{{tag> SymPhoTime software howto}}
-	{{tag> SPT64 SymPhoTime software howto}}	+
	~~TOC~~		~~TOC~~
	====== Configuring SymPhoTime64 ======		====== Configuring SymPhoTime64 ======

applications

Anonymous (anonymous@undisclosed.example.com) — 2026-01-12T10:25:53+00:00

2023/02/23 09:12		current
Line 53:		Line 53:

	[[howto:antibunching_measurements\|Photon antibunching measurements]]		[[howto:antibunching_measurements\|Photon antibunching measurements]]
		+
		+	[[howto:burst_duration_and_fcs_diffusion_time:burst_duration_and_fcs_diffusion_time\|Burst Duration and FCS Diffusion Time]]


Line 62:		Line 64:

	[[howto:calculate_ratiometric_single_pair_fret_distributions\|Calculate ratiometric single pair FRET distributions]]		[[howto:calculate_ratiometric_single_pair_fret_distributions\|Calculate ratiometric single pair FRET distributions]]
		+
		+	[[howto:burst_duration_and_fcs_diffusion_time:burst_duration_and_fcs_diffusion_time\|Burst Duration and FCS Diffusion Time]]

diamond_nv_centers

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

2018/04/19 14:11		current
Line 9:		Line 9:
	see also [[wp>Nitrogen-vacancy center]]		see also [[wp>Nitrogen-vacancy center]]

-	{{ howto:model_nv-centers.png\|Figure1: Model of the nitrogene vacancy in the diamond lattice}}	+	[{{ howto:model_nv-centers.png\|Figure1: Model of the nitrogene vacancy in the diamond lattice}}]

	==== Excitation ====		==== Excitation ====

fluorescence_lifetime_measurements_using_the_fluotime_300

Anonymous (anonymous@undisclosed.example.com) — 2016-09-08T01:19:58+00:00

2016/09/08 03:18		current
Line 34:		Line 34:
	{{ youtube>JLiDnlS5BS8?large }}		{{ youtube>JLiDnlS5BS8?large }}

-	This video demonstrates the special software application wizard of the FluoTime 300 system software for quantum yield measurements of Rhodamine 6G using an integrating sphere. The wizard guides the inexperienced user through all necessary steps to acquire high quality measurements.	+	This video demonstrates the special software application wizard of the FluoTime 300 system software for quantum yield measurements of Rhodamine 6G using an integrating sphere. The wizard guides the user through all necessary steps to acquire high quality measurements.

how_to_reinstall_symphotime

Anonymous (anonymous@undisclosed.example.com) — 2025-04-09T10:56:18+00:00

2025/04/09 12:55		current
Line 3:		Line 3:

	Updating from Windows 10 to Windows 11 should work smoothly, and all software should stay as it is. However it is a good idea to back-up the files mentioned below.		Updating from Windows 10 to Windows 11 should work smoothly, and all software should stay as it is. However it is a good idea to back-up the files mentioned below.
		+
	Please note: The Framegrabber MOU camera is not compatible with Windows 11 Windows 11 works with the newer USB MOU camera.		Please note: The Framegrabber MOU camera is not compatible with Windows 11 Windows 11 works with the newer USB MOU camera.
	==== Getting everything from the old computer ====		==== Getting everything from the old computer ====

measurement_hardware_instrumentation

Anonymous (anonymous@undisclosed.example.com) — 2018-06-25T07:18:07+00:00

2018/06/25 09:17		current
Line 32:		Line 32:
	[[video_tutorials#Kurt Thorn: Optical Sectioning and Confocal Microscopy\|Confocal microscoy and optical sectioning by Kurt Thorn]]		[[video_tutorials#Kurt Thorn: Optical Sectioning and Confocal Microscopy\|Confocal microscoy and optical sectioning by Kurt Thorn]]

-	[[technical docs:Beampath of the Zeiss LSM880]]	+	[[Beampath of the Zeiss LSM880]]

	[[howto:check_overlap_of_different_color_confocal_volumes\|How to check the overlap of different color confocal volumes]]		[[howto:check_overlap_of_different_color_confocal_volumes\|How to check the overlap of different color confocal volumes]]

calculate_and_fit_fcs_traces_with_the_fcs_script

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

2014/06/18 11:22		current
Line 17:		Line 17:
	* The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see http://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf ]. This list is extensive, but does not claim to be complete.		* The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see http://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf ]. This list is extensive, but does not claim to be complete.
	* Note that the diffusion coefficients of all dyes are temperature dependent.		* Note that the diffusion coefficients of all dyes are temperature dependent.
-	* FCS measurements are usually point measurements acquired with very sensitive detectors (typically SPAD or Hybrid PMT detectors).	+	* FCS measurements are usually point measurements acquired with very sensitive detectors (typically [[glossary:SPAD]] or [[glossary:Hybrid PMT]] detectors).
	* FCS is a technique that can be used over a wide range of concentrations, but ideal FCS concentrations are usually between 1 nM and 20 nM.		* FCS is a technique that can be used over a wide range of concentrations, but ideal FCS concentrations are usually between 1 nM and 20 nM.

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

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

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

using_the_antibunching_script

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

2014/06/19 09:36		current
Line 1:		Line 1:
	{{tag> antibunching howto tutorial correlation analysis SymPhoTime}}		{{tag> antibunching howto tutorial correlation analysis SymPhoTime}}
-	~~TOC~~	+	~~NOTOC~~

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

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

using_the_flcs_script_for_spectral_crosstalk_removal_via_flccs

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

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

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

pile-up_effect

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

2015/06/03 17:09		current
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-	{{tag>pile-up TCSPC pile-up dead_time}}	+	{{tag> TCSPC pile-up dead_time}}
	~~TOC~~		~~TOC~~

supported_mt200_pc_configuration_for_symphotime_32

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

2015/10/28 10:37		current
Line 1:		Line 1:
-	{{tag> SymPhoTime MT200 MicroTime SPT32}}	+	{{tag> SymPhoTime MT200 MicroTime SPT32 PC}}
	~~TOC~~		~~TOC~~

old_dongles_drivers_for_fluofit_symphotime_32

Anonymous (anonymous@undisclosed.example.com) — 2025-03-14T10:04:27+00:00

2025/03/14 11:02		current
Line 40:		Line 40:
	Our team successfully tested this setup using VMware Workstation Player 15.5.1, where both FluoFit and SPT32 dongles worked on an emulated Windows 7 (x64). Consider the following points:		Our team successfully tested this setup using VMware Workstation Player 15.5.1, where both FluoFit and SPT32 dongles worked on an emulated Windows 7 (x64). Consider the following points:

-	You will need valid licenses for both Windows 10/8 (host system) and Windows 7 (guest system).	+	You will need valid licenses for both newer Windows version (host system) and Windows 7 (guest system).

	VMware Workstation Player is free for non-commercial use, but the VM setup must be done by the user.		VMware Workstation Player is free for non-commercial use, but the VM setup must be done by the user.

how_to_work_with_the_instrument_response_function_irf

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

2021/03/23 11:49		current
Line 1:		Line 1:
-	{{tag>howto IRF tutorial analysis MT LSM_upgrade}}	+	{{tag>howto IRF tutorial analysis MicroTime LSM_upgrade}}

start

Anonymous (anonymous@undisclosed.example.com) — 2021-06-17T14:47:30+00:00

2020/05/04 14:11		current
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-	<WRAP center round box 60%>
-	===== News from the community=====
-	* 29.04.20 [[https://www.fret.community/announcement-protein-folding-and-dynamics-webinar/\|Protein folding and dynamics Webinar-Series.]]
-	* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\
-	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Exciting fundamental enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. \\ \\
-
-
-
-	[[general:community_news\|...see more...]]
-	</WRAP>

antibunching_measurements

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

2025/04/09 16:00		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.	+	Antibunching Measurements are (normally) measured in T2 mode. In T2 mode, all input channels of the TCSPC electronics are treated the same, also the "Sync" channel. Therefore the sync has to be disabled either by disconneting the cable, or by changing the CFD level or trigger level to such a value that the sync is not recognized any more. i.e. Sync rate = 0 . Directly in the Sepia software, the sync out can also be disabled.

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

calculate_fccs_trace_with_the_grouped_fcs_script

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

2015/02/25 15:01		current
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 =====

calculate_ratiometric_fret-images

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

2014/06/18 11:24		current
Line 3:		Line 3:
	~~NOTOC~~		~~NOTOC~~

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

calibrate_the_confocal_volume_for_fcs_using_the_fcs_calibration_script

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

2019/02/07 13:13		current
Line 17:		Line 17:
	Note: Calibration is a necessary task for FCS measurements. In a calibration procedure, the confocal volume VEff as well as the structural parameter κ (the ratio of z to xy extension) is determined using a reference dye. This is the prerequisite to determine correct diffusion coefficients and concentrations for the sample of interest.\\		Note: Calibration is a necessary task for FCS measurements. In a calibration procedure, the confocal volume VEff as well as the structural parameter κ (the ratio of z to xy extension) is determined using a reference dye. This is the prerequisite to determine correct diffusion coefficients and concentrations for the sample of interest.\\
	ATTO488 (ATTO-Tec, Germany) is a suited dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection around 500 – 540 nm.\\		ATTO488 (ATTO-Tec, Germany) is a suited dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection around 500 – 540 nm.\\
-	The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see [[https://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf]]]. This list is extensive, but does not claim to be complete.\\	+	The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant (see [[https://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf]]). This list is extensive, but does not claim to be complete.\\
	Note that the diffusion coefficients of all dyes are temperature dependent.\\		Note that the diffusion coefficients of all dyes are temperature dependent.\\
	FCS measurements are usually point measurements acquired with very sensitive detectors ([[glossary:spad\|SPAD]] or [[glossary:Hybrid PMT]] detectors).\\		FCS measurements are usually point measurements acquired with very sensitive detectors ([[glossary:spad\|SPAD]] or [[glossary:Hybrid PMT]] detectors).\\

determination_of_the_focal_width_with_the_focal

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

2014/06/18 13:00		current
Line 52:		Line 52:

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

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

pattern_matching

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

2014/05/23 09:39		current
Line 10:		Line 10:

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

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

using_the_anisotropy_image_script

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

2018/04/12 12:26		current
Line 52:		Line 52:

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

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

symphotime64

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

2014/05/23 09:34		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]].

interfacing_time_resolved_spectrometer_fluotime_300_microscope_microtime_100

Anonymous (anonymous@undisclosed.example.com) — 2020-05-18T14:19:46+00:00

2020/05/18 16:19		current
Line 1:		Line 1:
-	{{tag> video FT300 easyTau MT100 FLIM microscopy howto tutorial draft}}	+	{{tag> video FT300 easyTau MT100 FLIM microscopy howto tutorial}}

	====== Interfacing a time-resolved spectrometer (FluoTime 300) with a microscope (MicroTime 100) ======		====== Interfacing a time-resolved spectrometer (FluoTime 300) with a microscope (MicroTime 100) ======

registering_new_scripts

Anonymous (anonymous@undisclosed.example.com) — 2015-02-25T13:57:29+00:00

2014/06/18 17:46		current
Line 3:		Line 3:
	~~NOTOC~~		~~NOTOC~~

-	====== Registering new scripts in the SymPhoTime 64 ======	+	====== Registering New Scripts in the SymPhoTime 64 ======

tcspc_external_markers

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

2020/09/14 18:01		current
Line 31:		Line 31:
	* PI Controller: E725		* PI Controller: E725
	* PI Controller: E710 \\		* PI Controller: E710 \\
-	* Controller: E727 \\	+	* PI Controller: E727 \\
	* Controller: C867.2U2 (PI wide range scanner)		* Controller: C867.2U2 (PI wide range scanner)
	* Controller: C867.262 (PI wide range scanner)		* Controller: C867.262 (PI wide range scanner)

video_tutorials

Anonymous (anonymous@undisclosed.example.com) — 2014-11-17T16:36:53+00:00

2014/06/18 12:02		current
Line 31:		Line 31:
	[[http://www.ibiology.org/ibioseminars/techniques/kurt-thorn-part-1.html]]		[[http://www.ibiology.org/ibioseminars/techniques/kurt-thorn-part-1.html]]

-	=== Kurt Thorn: Two Photon Microscopy ===	+	==== Kurt Thorn: Two Photon Microscopy ====
	Kurt Thorn of UCSF talks about two-photon microscopy which uses intense pulsed lasers to image deep into biological samples. It can be used for imaging thick tissue specimens or even imaging inside of live animals.		Kurt Thorn of UCSF talks about two-photon microscopy which uses intense pulsed lasers to image deep into biological samples. It can be used for imaging thick tissue specimens or even imaging inside of live animals.

Line 39:		Line 39:


-	=== Philippe Bastiaens: Fluorescence Lifetime Microscopy (FLIM) ===	+	==== Philippe Bastiaens: Fluorescence Lifetime Microscopy (FLIM) ====
	Fluorescence-lifetime imaging microscopy or [[glossary:flim\|FLIM]] is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography.		Fluorescence-lifetime imaging microscopy or [[glossary:flim\|FLIM]] is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography.
	The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample ([[wp>Fluorescence-lifetime_imaging_microscopy \| FLIM]]).		The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample ([[wp>Fluorescence-lifetime_imaging_microscopy \| FLIM]]).

bifl

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

2014/04/09 22:38		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 [[products: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.

differential_count_rate

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

2017/09/24 05:34		current
Line 17:		Line 17:
	Not really. In case of pulsed signals the average count rate is a misleading quantity. An average count rate value does not take into account when and how those photons are emitted and detected. Interpreting a 100 kcps intensity as a constant emission rate (Poisson mean rate, in math terms) is a misconception. The physics of the measurement is completely different. These photons are obviously not emitted evenly, one by one over the whole one second period. They arrive to the detector bunched, as flashes. These are short time intervals with huge photon density (rate), separated by long "dark", quiet periods.		Not really. In case of pulsed signals the average count rate is a misleading quantity. An average count rate value does not take into account when and how those photons are emitted and detected. Interpreting a 100 kcps intensity as a constant emission rate (Poisson mean rate, in math terms) is a misconception. The physics of the measurement is completely different. These photons are obviously not emitted evenly, one by one over the whole one second period. They arrive to the detector bunched, as flashes. These are short time intervals with huge photon density (rate), separated by long "dark", quiet periods.

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

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

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

fast_lifetime

Anonymous (anonymous@undisclosed.example.com) — 2013-08-06T14:43:32+00:00

2013/08/06 16:43		current
Line 3:		Line 3:
	A method for estimating the average lifetime, calculated non-distinctive on different exponentials in a simple single path calculation, compared to a complete fitting operation against a more complex, non-linear model. Often used with [[FLIM]] go get a first idea of the lifetime distribution in the image.		A method for estimating the average lifetime, calculated non-distinctive on different exponentials in a simple single path calculation, compared to a complete fitting operation against a more complex, non-linear model. Often used with [[FLIM]] go get a first idea of the lifetime distribution in the image.

-	In detail, FastLT is calculating the barycentre of the (pseudo–)pixel's decay. The time span from the barycentre of the IRF to the barycentre of the decay equals the average lifetime. This estimate is very fast and does not suffer as much from low statistics. If an [[IRF]] is not available, the "time zero" $t_0_$ has to be estimated differently, for example by using the rising flank of the decay or the entrance of the [[FWHM]] interval. However, this may introduce a systematic shift in the estimated average lifetimes.	+	In detail, FastLT is calculating the barycentre of the (pseudo–)pixel's decay. The time span from the barycentre of the IRF to the barycentre of the decay equals the average lifetime. This estimate is very fast and does not suffer as much from low statistics. If an [[IRF]] is not available, the "time zero" $t_\theta$ has to be estimated differently, for example by using the rising flank of the decay or the entrance of the [[FWHM]] interval. However, this may introduce a systematic shift in the estimated average lifetimes.

tttr

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

2015/02/10 17:42		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 [[glossary:T3-mode]] 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.

lifetime_component_decomposition_phasor_plot_analysis

Anonymous (anonymous@undisclosed.example.com) — 2025-06-16T07:17:47+00:00

2025/06/16 09:17		current
Line 11:		Line 11:

	* Sample information:		* Sample information:
-	* Daisy Pollen	+	* Daisy pollen
		+
		+	---

	* Software information: NIS-Elements version 5.42.06 with PicoQuant FLIM-FCS full integration		* Software information: NIS-Elements version 5.42.06 with PicoQuant FLIM-FCS full integration
	{{:lifetime_component_decomposition_in_phasor_plot.mp4?1000\|}}		{{:lifetime_component_decomposition_in_phasor_plot.mp4?1000\|}}
-		+
	* Sample information:		* Sample information:
	* Glial Fibrillary Acidic Protein (GFAP) — labeled with BDP-TMR		* Glial Fibrillary Acidic Protein (GFAP) — labeled with BDP-TMR
	* Mitochondria (via TOM20) — labeled with CY3		* Mitochondria (via TOM20) — labeled with CY3

dead_time

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

2015/06/03 17:04		current
Line 2:		Line 2:
	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.		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]].	+	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.		Note that not only TCSPC devices but also photon counting devices exhibit dead times.

mcs

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

2014/01/21 17:38		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.

symphotime_32

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

2026/04/23 19:16	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]]

phasor_plot_structure_separation

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

2025/06/16 10:36		current
Line 1:		Line 1:
	{{tag> FLIM acquisition Nikon AX Phasor video LSM_upgrade howto}}		{{tag> FLIM acquisition Nikon AX Phasor video LSM_upgrade howto}}

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

	Introduction		Introduction

start

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

2025/02/14 10:43		current
Line 10:		Line 10:
	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:

flim

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:28:34+00:00

2015/11/03 15:28		current
Line 1:		Line 1:
-	~~TOC~~	+
	{{tag>FLIM}}		{{tag>FLIM}}
		+
		+	~~TOC~~
	====== FLIM ======		====== FLIM ======

marquardt-levenberg

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:34:33+00:00

2014/04/09 22:38		current
Line 1:		Line 1:
	====== Marquardt-Levenberg ======		====== Marquardt-Levenberg ======

-	The Marquardt-Levenberg method is a means of optimising parameters in a least squares fit. It is used in our [[products:FluoFit]], [[products:SymPhoTime]] and a variety of other products (online-Fitting, FluoLib...).	+	The Marquardt-Levenberg method is a means of optimising parameters in a least squares fit. It is used in our [[software:FluoFit]], [[software:SymPhoTime]] and a variety of other products (online-Fitting, FluoLib...).

	Basically it works by examining how changes of a certain parameter affect the residuals trace, that means, it calculates a pointwise gradient of the residuals trace for each parameter. Marquard-Levenberg optimisation is fairly efficient and is regarded as state-of-the-art in least squares fitting.		Basically it works by examining how changes of a certain parameter affect the residuals trace, that means, it calculates a pointwise gradient of the residuals trace for each parameter. Marquard-Levenberg optimisation is fairly efficient and is regarded as state-of-the-art in least squares fitting.

align_beam_backreflection

Anonymous (anonymous@undisclosed.example.com) — 2022-08-29T11:17:12+00:00

2022/08/29 13:15		current
Line 12:		Line 12:
	Adding a drop of water on top of the coverslip reduces the back-scattering intensity by an order of magnitude or so, but the basic shape remains the same. Even if one removes everything extra from the beam path, these fringes remain there, they are universal. Note that all those images above are slightly defocused.		Adding a drop of water on top of the coverslip reduces the back-scattering intensity by an order of magnitude or so, but the basic shape remains the same. Even if one removes everything extra from the beam path, these fringes remain there, they are universal. Note that all those images above are slightly defocused.

-	Such images are used for the daily and fundamental alignment of the MicroTime 200. The detailed procedure is described in the user manual of MicroTime 200. Although the MicroTime is extremely robust we suggest to spend 5-10 minutes every day for verifying the quality of the alignment by inspecting the back-reflection pattern. Have a look how this is used also by watching the tutorial [[howto:exchange_dichroic_mt200\|How to exchange the main dichroic of the MicroTime 200.]]	+	Such images are used for daily and fundamental alignment of the MicroTime 200. A detailed procedure is described in the user manual of MicroTime 200. Although MicroTime is extremely robust, we suggest to spend 5-10 minutes every day for verifying the quality of the alignment by inspecting the back-reflection pattern. Have a look how this is used also by watching the tutorial [[howto:exchange_dichroic_mt200\|How to exchange the main dichroic of the MicroTime 200.]]

	The paper by [[ http://rspa.royalsocietypublishing.org/content/253/1274/358\|B. Richards and E. Wolf: Electromagnetic diffraction in optical systems, II. Structure of the image field in an aplanatic system]] shows such 4 lobed patterns for polarized light.		The paper by [[ http://rspa.royalsocietypublishing.org/content/253/1274/358\|B. Richards and E. Wolf: Electromagnetic diffraction in optical systems, II. Structure of the image field in an aplanatic system]] shows such 4 lobed patterns for polarized light.

	Similar figures can be found in this [[https://books.google.cz/books?id=kURdAAAAQBAJ\|classic textbook]].		Similar figures can be found in this [[https://books.google.cz/books?id=kURdAAAAQBAJ\|classic textbook]].

measuring_quantum_yield

Anonymous (anonymous@undisclosed.example.com) — 2017-07-12T13:16:44+00:00

2017/07/12 15:13		current
Line 8:		Line 8:
	{{ youtube>JLiDnlS5BS8?large }}		{{ youtube>JLiDnlS5BS8?large }}

-	===== Setp-by-Step =====	+	===== Step-by-Step =====
-	* Insert the integrating sphere with caution in order not to hit any optical elements in the FluoTime 300. The integrating sphere assembly has an indexed lid to ensure proper orientation.	+	* Mount the integrating sphere carefully. Make sure you do not hit any optical elements inside the FluoTime 300. The integrating sphere assembly has an indexed lid to ensure proper orientation.

	{{ :howto:2013-12-19_at_14-30-03.jpg?600 \|}}		{{ :howto:2013-12-19_at_14-30-03.jpg?600 \|}}

phasor_analysis

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

2019/08/28 18:15		current
Line 1:		Line 1:
-	{{tag> FLIM howto phasor bin-export binexport analysis tutorial SymPhoTime Globals software}}	+	{{tag> FLIM howto phasor bin-export bin_export analysis tutorial SymPhoTime Globals software}}

	~~TOC~~		~~TOC~~

t3r_antibunching_-_slow_decay

Anonymous (anonymous@undisclosed.example.com) — 2022-02-01T11:32:53+00:00

2022/02/01 11:54		current
Line 7:		Line 7:

	==== A decay caused by correlation statistics ====		==== A decay caused by correlation statistics ====
		+
		+	[{{howto:slope.png ?478x300\|Fig. 1: Baseline decay }}]
		+
	This decay is caused by correlation statistics. This happens because you correlate the first arriving photon against a photon at the longer time, which will be an increasingly unlikely event because the earliest stop photons always win and the experiment restarts.		This decay is caused by correlation statistics. This happens because you correlate the first arriving photon against a photon at the longer time, which will be an increasingly unlikely event because the earliest stop photons always win and the experiment restarts.

-	I attached a simulation.	+	A simulation with 90ns dead-time period:

-	[{{howto:logf.png?320x240\|Fig. 1: Logfile logf.png }}]	+	[{{howto:logf.png?320x240\|Fig. 2: Simulation }}]

supported_mt200_pc_configuration_for_symphotime_64

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

2023/02/17 13:44		current
Line 1:		Line 1:
-	{{tag>SymPhoTime MicroTime200 MicroTime100 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~~

symphotime

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

2015/02/10 17:32		current
Line 3:		Line 3:
	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.		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:SymPhoTim64]].	+	After several very successful years [[software:SymPhoTime32]] has been replaced by the completely rewritten 64bit version [[software:SymPhoTime64]].

laser_intensity_response

Anonymous (anonymous@undisclosed.example.com) — 2025-03-11T08:23:49+00:00

2025/03/11 09:23		current
Line 1:		Line 1:
	{{tag>FAQ LDH PLS PDL Laser SymPhoTime SPT SEPIA}}		{{tag>FAQ LDH PLS PDL Laser SymPhoTime SPT SEPIA}}

-	====== The intensity response of my laser is not linear ======	+	====== The Intensity Response of my Laser is not linear ======
	or		or
-	====== My laser does not turn on with low power ======	+	====== My Laser does not turn on with low Power ======

	===== Background =====		===== Background =====

the_dark_counts_of_my_spads_increased._when_do_i_need_to_take_action

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T15:46:14+00:00

2025/01/31 09:57		current
Line 1:		Line 1:
	====== The Dark Counts of my SPADs increased. When do I need to take action? ======		====== The Dark Counts of my SPADs increased. When do I need to take action? ======
-	{{tag> detector faq SPAD dcps dark_counts}}	+	{{tag> detectors faq SPAD dcps dark_counts}}

	SPAD detectors (Excelitas SPCMs or MPD PDMs) are relatively robust devices, but they can exhibit ageing, which shows mostly in the level of dark counts. Dark counts are the count rate on the detector when no light at all can get to the active area of the detector.		SPAD detectors (Excelitas SPCMs or MPD PDMs) are relatively robust devices, but they can exhibit ageing, which shows mostly in the level of dark counts. Dark counts are the count rate on the detector when no light at all can get to the active area of the detector.

advantages_and_disadvantages_of_two_photon_excitation_tpe

Anonymous (anonymous@undisclosed.example.com) — 2014-01-28T13:07:58+00:00

2014/01/28 14:07		current
Line 1:		Line 1:
-	{{tag>TPE}}
	====== Advantages and disadvantages of two photon excitation (TPE) ======		====== Advantages and disadvantages of two photon excitation (TPE) ======

community_news

Anonymous (anonymous@undisclosed.example.com) — 2020-05-04T12:11:21+00:00

2020/04/29 10:35		current
Line 4:		Line 4:
	* 29.04.20 [[https://www.fret.community/announcement-protein-folding-and-dynamics-webinar/\|Protein folding and dynamics Webinar-Series.]] \\ \\		* 29.04.20 [[https://www.fret.community/announcement-protein-folding-and-dynamics-webinar/\|Protein folding and dynamics Webinar-Series.]] \\ \\
	* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\		* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\
-	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Fundamendal enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. The gates were built from small, designed proteins that all have a similar structure but where one module can be designed to interact specifically with another module. Using monomers and covalently connected monomers as inputs and encoding specificity through designed hydrogen-bond networks allowed the construction of two-input or three-input gates based on competitive binding. \\ \\	+	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Fundamental enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. The gates were built from small, designed proteins that all have a similar structure but where one module can be designed to interact specifically with another module. Using monomers and covalently connected monomers as inputs and encoding specificity through designed hydrogen-bond networks allowed the construction of two-input or three-input gates based on competitive binding. \\ \\
	* 31.03.20 FRET community has a new advisory board, [[https://www.fret.community/\|FRET community]]. Happy that Dr. Marcelle König , a senior scientist in PicoQuant was elected. \\ \\		* 31.03.20 FRET community has a new advisory board, [[https://www.fret.community/\|FRET community]]. Happy that Dr. Marcelle König , a senior scientist in PicoQuant was elected. \\ \\
	* 31.03.20 Extremely helpful reference paper about confocal microscopy. [[https://www.nature.com/articles/s41596-020-0313-9\|Tutorial: guidance for quantitative confocal microscopy]]. Available as a poster [[https://www.nature.com/articles/s41596-020-0307-7\|here.]] \\ \\		* 31.03.20 Extremely helpful reference paper about confocal microscopy. [[https://www.nature.com/articles/s41596-020-0313-9\|Tutorial: guidance for quantitative confocal microscopy]]. Available as a poster [[https://www.nature.com/articles/s41596-020-0307-7\|here.]] \\ \\

deconvolution

Anonymous (anonymous@undisclosed.example.com) — 2013-08-06T14:41:19+00:00

2013/08/06 16:13		current
Line 1:		Line 1:
	====== Deconvolution ======		====== Deconvolution ======
-
-	===== What is "Deconvolution"? =====

	Deconvolution is used in time domain data analysis for removal of broadening effects due to instrumental resolution. In this context deconvolution is mainly concerned with the [[IRF]] (or lamp function) including the finite light source pulse width and other broadening effects (e.g. electronics). The effects caused by the [[IRF]] are dominant in the onset of a decay curve.		Deconvolution is used in time domain data analysis for removal of broadening effects due to instrumental resolution. In this context deconvolution is mainly concerned with the [[IRF]] (or lamp function) including the finite light source pulse width and other broadening effects (e.g. electronics). The effects caused by the [[IRF]] are dominant in the onset of a decay curve.
Line 8:		Line 6:
	===== How deconvolution should be done =====		===== How deconvolution should be done =====

-	Best, not at all ;-). At PQ we use [[reconvolution]] instead, for a variety of good reasons (see [[IRF]]). If one cannot resist applying deconvolution, the best way of doing it (at least for time domain data) would be via Fourier transform. Fourier transform is a slow process (even with [[FFT]]) and it has to be done twice, since the deconvoultion itself is performed on the transformed data set, which has to be retransformed afterwards.	+	Best, not at all ;-). At PicoQuant, for a variety of good reasons (see [[IRF]]) we use [[reconvolution]] instead. If one cannot resist applying deconvolution, the best way of doing it (at least for time domain data) would be via Fourier transform. Fourier transform is a slow process (even with [[FFT]]) and it has to be done twice, since the deconvoultion itself is performed on the transformed data set, which has to be retransformed afterwards.


Line 14:		Line 12:

	The most prominent application of deconvolution techniques is imaging. Usually one tries to remove blurring by deconvolution (or deconvolution-like) techniques. What holds for time domain data, also holds for imaging: Deconvolution has a disturbingly high potential for producing artefacts - and there is no way of telling apart artefacts and effects. The images may look nicer (which undoubtedly is valuable especially for publication purposes), but the content of information has not necessarily improved.		The most prominent application of deconvolution techniques is imaging. Usually one tries to remove blurring by deconvolution (or deconvolution-like) techniques. What holds for time domain data, also holds for imaging: Deconvolution has a disturbingly high potential for producing artefacts - and there is no way of telling apart artefacts and effects. The images may look nicer (which undoubtedly is valuable especially for publication purposes), but the content of information has not necessarily improved.
-

fast_flim

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:29:58+00:00

2013/08/06 15:49		current
Line 1:		Line 1:
		+	{{tag>FLIM fast_flim}}
	====== Fast FLIM ======		====== Fast FLIM ======

	[[FLIM]] image, using the [[fast lifetime]] calculation. The color-coded fast lifetime of each pixel represents the average photon arrival time with respect to the preceding laser pulse.		[[FLIM]] image, using the [[fast lifetime]] calculation. The color-coded fast lifetime of each pixel represents the average photon arrival time with respect to the preceding laser pulse.

		+	===== FLIM in this wiki =====

		+	{{topic>FLIM -open_source&nodate&nouser}}

tcspc

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

2014/01/28 14:06		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.	+	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.

check_overlap_of_different_color_confocal_volumes

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

2014/01/30 17:02		current
Line 1:		Line 1:
	{{tag>howto confocal_volume}}		{{tag>howto confocal_volume}}
-	====== How to check the overlap of different color confocal volumes ======	+	====== How to Check the Overlap of Different Color Confocal Volumes ======

	Because both lasers are fed through the same fiber, there cannot be any optical beam displacement. According to my records, your system has the latest apochromatic fiber outcoupler, therefore there is practically nothing you can improve: lasers mounted in the [[products:LCU]] are always well overlapped. This is a simple consequence of the [[Products:MicroTime 200]] design.		Because both lasers are fed through the same fiber, there cannot be any optical beam displacement. According to my records, your system has the latest apochromatic fiber outcoupler, therefore there is practically nothing you can improve: lasers mounted in the [[products:LCU]] are always well overlapped. This is a simple consequence of the [[Products:MicroTime 200]] design.

exchange_dichroic_mt200

Anonymous (anonymous@undisclosed.example.com) — 2023-02-17T12:42:54+00:00

2021/06/25 11:19		current
Line 1:		Line 1:
-		+	{{tag>howto MicroTime200 dichroic microscopy}}
-	{{tag>howto MicroTime200 dichroic microscopy MT200}}	+

	====== How to exchange the main dichroic of the MicroTime 200 ======		====== How to exchange the main dichroic of the MicroTime 200 ======

start

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

2024/12/19 16:57		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 -draft &nodate&nouser}}

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

hydraharp_400

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

2015/09/23 13:43		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 =====

howto

Anonymous (anonymous@undisclosed.example.com) — 2026-01-12T10:03:28+00:00

2025/06/16 10:38		current
Line 1:		Line 1:
	[[howto:phasor_plot_structure_separation:phasor_plot_structure_separation\|Phasor-Based Structural Separation Using FLIM]]		[[howto:phasor_plot_structure_separation:phasor_plot_structure_separation\|Phasor-Based Structural Separation Using FLIM]]
-

	[[howto:AX_FLIM_Examples:AX_FLIM_Examples\|NIS_Element_based_FLIM_Measurement_Examples]]		[[howto:AX_FLIM_Examples:AX_FLIM_Examples\|NIS_Element_based_FLIM_Measurement_Examples]]
		+
		+	[[howto:Burst Duration and FCS Diffusion Time:Burst Duration and FCS Diffusion Time\|Burst Duration and FCS Diffusion Time]]

syntax

Anonymous (anonymous@undisclosed.example.com) — 2026-04-23T08:17:27+00:00

2026/04/23 19:16	current
Line 1:	Line 1:
	+	====== Formatting Syntax ======
	+
	+	[[doku>DokuWiki]] supports some simple markup language, which tries to make the datafiles to be as readable as possible. This page contains all possible syntax you may use when editing the pages. Simply have a look at the source of this page by pressing "Edit this page". If you want to try something, just use the [[playground:playground\|playground]] page. The simpler markup is easily accessible via [[doku>toolbar\|quickbuttons]], too.
	+
	+	===== Basic Text Formatting =====
	+
	+	DokuWiki supports bold, //italic//, __underlined__ and ''monospaced'' texts. Of course you can __//''combine''//__ all these.
	+
	+	DokuWiki supports bold, //italic//, __underlined__ and ''monospaced'' texts.
	+	Of course you can __//''combine''//__ all these.
	+
	+	You can use <sub>subscript</sub> and <sup>superscript</sup>, too.
	+
	+	You can use <sub>subscript</sub> and <sup>superscript</sup>, too.
	+
	+	You can mark something as <del>deleted</del> as well.
	+
	+	You can mark something as <del>deleted</del> as well.
	+
	+	Paragraphs are created from blank lines. If you want to force a newline without a paragraph, you can use two backslashes followed by a whitespace or the end of line.
	+
	+	This is some text with some linebreaks\\ Note that the
	+	two backslashes are only recognized at the end of a line\\
	+	or followed by\\ a whitespace \\this happens without it.
	+
	+	This is some text with some linebreaks\\ Note that the
	+	two backslashes are only recognized at the end of a line\\
	+	or followed by\\ a whitespace \\this happens without it.
	+
	+	You should use forced newlines only if really needed.
	+
	+	===== Links =====
	+
	+	DokuWiki supports multiple ways of creating links.
	+
	+	==== External ====
	+
	+	External links are recognized automagically: http://www.google.com or simply www.google.com - You can set the link text as well: [[http://www.google.com\|This Link points to google]]. Email addresses like this one: <andi@splitbrain.org> are recognized, too.
	+
	+	DokuWiki supports multiple ways of creating links. External links are recognized
	+	automagically: http://www.google.com or simply www.google.com - You can set
	+	link text as well: [[http://www.google.com\|This Link points to google]]. Email
	+	addresses like this one: <andi@splitbrain.org> are recognized, too.
	+
	+	==== Internal ====
	+
	+	Internal links are created by using square brackets. You can either just give a [[pagename]] or use an additional [[pagename\|link text]].
	+
	+	Internal links are created by using square brackets. You can either just give
	+	a [[pagename]] or use an additional [[pagename\|link text]].
	+
	+	[[doku>pagename\|Wiki pagenames]] are converted to lowercase automatically, special characters are not allowed.
	+
	+	You can use [[some:namespaces]] by using a colon in the pagename.
	+
	+	You can use [[some:namespaces]] by using a colon in the pagename.
	+
	+	For details about namespaces see [[doku>namespaces]].
	+
	+	Linking to a specific section is possible, too. Just add the section name behind a hash character as known from HTML. This links to [[syntax#internal\|this Section]].
	+
	+	This links to [[syntax#internal\|this Section]].
	+
	+	Notes:
	+
	+	* Links to [[syntax\|existing pages]] are shown in a different style from [[nonexisting]] ones.
	+	* DokuWiki does not use [[wp>CamelCase]] to automatically create links by default, but this behavior can be enabled in the [[doku>config]] file. Hint: If DokuWiki is a link, then it's enabled.
	+	* When a section's heading is changed, its bookmark changes, too. So don't rely on section linking too much.
	+
	+	==== Interwiki ====
	+
	+	DokuWiki supports [[doku>Interwiki]] links. These are quick links to other Wikis. For example this is a link to Wikipedia's page about Wikis: [[wp>Wiki]].
	+
	+	DokuWiki supports [[doku>Interwiki]] links. These are quick links to other Wikis.
	+	For example this is a link to Wikipedia's page about Wikis: [[wp>Wiki]].
	+
	+	==== Windows Shares ====
	+
	+	Windows shares like [[\\server\share\|this]] are recognized, too. Please note that these only make sense in a homogeneous user group like a corporate [[wp>Intranet]].
	+
	+	Windows Shares like [[\\server\share\|this]] are recognized, too.
	+
	+	Notes:
	+
	+	* For security reasons direct browsing of windows shares only works in Microsoft Internet Explorer per default (and only in the "local zone").
	+	* For Mozilla and Firefox it can be enabled through different workaround mentioned in the [[http://kb.mozillazine.org/Links_to_local_pages_do_not_work\|Mozilla Knowledge Base]]. However, there will still be a JavaScript warning about trying to open a Windows Share. To remove this warning (for all users), put the following line in ''conf/lang/en/lang.php'' (more details at [[doku>localization#changing_some_localized_texts_and_strings_in_your_installation\|localization]]): <code - conf/lang/en/lang.php>
	+	<?php
	+	/**
	+	* Customization of the english language file
	+	* Copy only the strings that needs to be modified
	+	*/
	+	$lang['js']['nosmblinks'] = '';
	+	</code>
	+
	+	==== Image Links ====
	+
	+	You can also use an image to link to another internal or external page by combining the syntax for links and [[#images_and_other_files\|images]] (see below) like this:
	+
	+	[[http://php.net\|{{wiki:dokuwiki-128.png}}]]
	+
	+	[[http://php.net\|{{wiki:dokuwiki-128.png}}]]
	+
	+	Please note: The image formatting is the only formatting syntax accepted in link names.
	+
	+	The whole [[#images_and_other_files\|image]] and [[#links\|link]] syntax is supported (including image resizing, internal and external images and URLs and interwiki links).
	+
	+	===== Footnotes =====
	+
	+	You can add footnotes ((This is a footnote)) by using double parentheses.
	+
	+	You can add footnotes ((This is a footnote)) by using double parentheses.
	+
	+	===== Sectioning =====
	+
	+	You can use up to five different levels of headlines to structure your content. If you have more than three headlines, a table of contents is generated automatically -- this can be disabled by including the string ''<nowiki>~~NOTOC~~</nowiki>'' in the document.
	+
	+	==== Headline Level 3 ====
	+	=== Headline Level 4 ===
	+	== Headline Level 5 ==
	+
	+	==== Headline Level 3 ====
	+	=== Headline Level 4 ===
	+	== Headline Level 5 ==
	+
	+	By using four or more dashes, you can make a horizontal line:
	+
	+	----
	+
	+	===== Media Files =====
	+
	+	You can include external and internal [[doku>images\|images, videos and audio files]] with curly brackets. Optionally you can specify the size of them.
	+
	+	Real size: {{wiki:dokuwiki-128.png}}
	+
	+	Resize to given width: {{wiki:dokuwiki-128.png?50}}
	+
	+	Resize to given width and height((when the aspect ratio of the given width and height doesn't match that of the image, it will be cropped to the new ratio before resizing)): {{wiki:dokuwiki-128.png?200x50}}
	+
	+	Resized external image: {{https://www.php.net/images/php.gif?200x50}}
	+
	+	Real size: {{wiki:dokuwiki-128.png}}
	+	Resize to given width: {{wiki:dokuwiki-128.png?50}}
	+	Resize to given width and height: {{wiki:dokuwiki-128.png?200x50}}
	+	Resized external image: {{https://www.php.net/images/php.gif?200x50}}
	+
	+
	+	By using left or right whitespaces you can choose the alignment.
	+
	+	{{ wiki:dokuwiki-128.png}}
	+
	+	{{wiki:dokuwiki-128.png }}
	+
	+	{{ wiki:dokuwiki-128.png }}
	+
	+	{{ wiki:dokuwiki-128.png}}
	+	{{wiki:dokuwiki-128.png }}
	+	{{ wiki:dokuwiki-128.png }}
	+
	+	Of course, you can add a title (displayed as a tooltip by most browsers), too.
	+
	+	{{ wiki:dokuwiki-128.png \|This is the caption}}
	+
	+	{{ wiki:dokuwiki-128.png \|This is the caption}}
	+
	+	For linking an image to another page see [[#Image Links]] above.
	+
	+	==== Supported Media Formats ====
	+
	+	DokuWiki can embed the following media formats directly.
	+
	+	\| Image \| ''gif'', ''jpg'', ''png'' \|
	+	\| Video \| ''webm'', ''ogv'', ''mp4'' \|
	+	\| Audio \| ''ogg'', ''mp3'', ''wav'' \|
	+	\| Flash \| ''swf'' \|
	+
	+	If you specify a filename that is not a supported media format, then it will be displayed as a link instead.
	+
	+	By adding ''?linkonly'' you provide a link to the media without displaying it inline
	+
	+	{{wiki:dokuwiki-128.png?linkonly}}
	+
	+	{{wiki:dokuwiki-128.png?linkonly}} This is just a link to the image.
	+
	+	==== Fallback Formats ====
	+
	+	Unfortunately not all browsers understand all video and audio formats. To mitigate the problem, you can upload your file in different formats for maximum browser compatibility.
	+
	+	For example consider this embedded mp4 video:
	+
	+	{{video.mp4\|A funny video}}
	+
	+	When you upload a ''video.webm'' and ''video.ogv'' next to the referenced ''video.mp4'', DokuWiki will automatically add them as alternatives so that one of the three files is understood by your browser.
	+
	+	Additionally DokuWiki supports a "poster" image which will be shown before the video has started. That image needs to have the same filename as the video and be either a jpg or png file. In the example above a ''video.jpg'' file would work.
	+
	+	===== Lists =====
	+
	+	Dokuwiki supports ordered and unordered lists. To create a list item, indent your text by two spaces and use a ''*'' for unordered lists or a ''-'' for ordered ones.
	+
	+	* This is a list
	+	* The second item
	+	* You may have different levels
	+	* Another item
	+
	+	- The same list but ordered
	+	- Another item
	+	- Just use indention for deeper levels
	+	- That's it
	+
	+	<code>
	+	* This is a list
	+	* The second item
	+	* You may have different levels
	+	* Another item
	+
	+	- The same list but ordered
	+	- Another item
	+	- Just use indention for deeper levels
	+	- That's it
	+	</code>
	+
	+	Also take a look at the [[doku>faq:lists\|FAQ on list items]].
	+
	+	===== Text Conversions =====
	+
	+	DokuWiki can convert certain pre-defined characters or strings into images or other text or HTML.
	+
	+	The text to image conversion is mainly done for smileys. And the text to HTML conversion is used for typography replacements, but can be configured to use other HTML as well.
	+
	+	==== Text to Image Conversions ====
	+
	+	DokuWiki converts commonly used [[wp>emoticon]]s to their graphical equivalents. Those [[doku>Smileys]] and other images can be configured and extended. Here is an overview of Smileys included in DokuWiki:
	+
	+	* 8-) %% 8-) %%
	+	* 8-O %% 8-O %%
	+	* :-( %% :-( %%
	+	* :-) %% :-) %%
	+	* =) %% =) %%
	+	* :-/ %% :-/ %%
	+	* :-\ %% :-\ %%
	+	* :-? %% :-? %%
	+	* :-D %% :-D %%
	+	* :-P %% :-P %%
	+	* :-O %% :-O %%
	+	* :-X %% :-X %%
	+	* :-\| %% :-\| %%
	+	* ;-) %% ;-) %%
	+	* ^_^ %% ^_^ %%
	+	* m( %% m( %%
	+	* :?: %% :?: %%
	+	* :!: %% :!: %%
	+	* LOL %% LOL %%
	+	* FIXME %% FIXME %%
	+	* DELETEME %% DELETEME %%
	+
	+	==== Text to HTML Conversions ====
	+
	+	Typography: [[DokuWiki]] can convert simple text characters to their typographically correct entities. Here is an example of recognized characters.
	+
	+	-> <- <-> => <= <=> >> << -- --- 640x480 (c) (tm) (r)
	+	"He thought 'It's a man's world'..."
	+
	+	<code>
	+	-> <- <-> => <= <=> >> << -- --- 640x480 (c) (tm) (r)
	+	"He thought 'It's a man's world'..."
	+	</code>
	+
	+	The same can be done to produce any kind of HTML, it just needs to be added to the [[doku>entities\|pattern file]].
	+
	+	There are three exceptions which do not come from that pattern file: multiplication entity (640x480), 'single' and "double quotes". They can be turned off through a [[doku>config:typography\|config option]].
	+
	+	===== Quoting =====
	+
	+	Some times you want to mark some text to show it's a reply or comment. You can use the following syntax:
	+
	+	<code>
	+	I think we should do it
	+
	+	> No we shouldn't
	+
	+	>> Well, I say we should
	+
	+	> Really?
	+
	+	>> Yes!
	+
	+	>>> Then lets do it!
	+	</code>
	+
	+	I think we should do it
	+
	+	> No we shouldn't
	+
	+	>> Well, I say we should
	+
	+	> Really?
	+
	+	>> Yes!
	+
	+	>>> Then lets do it!
	+
	+	===== Tables =====
	+
	+	DokuWiki supports a simple syntax to create tables.
	+
	+	^ Heading 1 ^ Heading 2 ^ Heading 3 ^
	+	\| Row 1 Col 1 \| Row 1 Col 2 \| Row 1 Col 3 \|
	+	\| Row 2 Col 1 \| some colspan (note the double pipe) \|\|
	+	\| Row 3 Col 1 \| Row 3 Col 2 \| Row 3 Col 3 \|
	+
	+	Table rows have to start and end with a ''\|'' for normal rows or a ''^'' for headers.
	+
	+	^ Heading 1 ^ Heading 2 ^ Heading 3 ^
	+	\| Row 1 Col 1 \| Row 1 Col 2 \| Row 1 Col 3 \|
	+	\| Row 2 Col 1 \| some colspan (note the double pipe) \|\|
	+	\| Row 3 Col 1 \| Row 3 Col 2 \| Row 3 Col 3 \|
	+
	+	To connect cells horizontally, just make the next cell completely empty as shown above. Be sure to have always the same amount of cell separators!
	+
	+	Vertical tableheaders are possible, too.
	+
	+	\| ^ Heading 1 ^ Heading 2 ^
	+	^ Heading 3 \| Row 1 Col 2 \| Row 1 Col 3 \|
	+	^ Heading 4 \| no colspan this time \| \|
	+	^ Heading 5 \| Row 2 Col 2 \| Row 2 Col 3 \|
	+
	+	As you can see, it's the cell separator before a cell which decides about the formatting:
	+
	+	\| ^ Heading 1 ^ Heading 2 ^
	+	^ Heading 3 \| Row 1 Col 2 \| Row 1 Col 3 \|
	+	^ Heading 4 \| no colspan this time \| \|
	+	^ Heading 5 \| Row 2 Col 2 \| Row 2 Col 3 \|
	+
	+	You can have rowspans (vertically connected cells) by adding ''%%:::%%'' into the cells below the one to which they should connect.
	+
	+	^ Heading 1 ^ Heading 2 ^ Heading 3 ^
	+	\| Row 1 Col 1 \| this cell spans vertically \| Row 1 Col 3 \|
	+	\| Row 2 Col 1 \| ::: \| Row 2 Col 3 \|
	+	\| Row 3 Col 1 \| ::: \| Row 2 Col 3 \|
	+
	+	Apart from the rowspan syntax those cells should not contain anything else.
	+
	+	^ Heading 1 ^ Heading 2 ^ Heading 3 ^
	+	\| Row 1 Col 1 \| this cell spans vertically \| Row 1 Col 3 \|
	+	\| Row 2 Col 1 \| ::: \| Row 2 Col 3 \|
	+	\| Row 3 Col 1 \| ::: \| Row 2 Col 3 \|
	+
	+	You can align the table contents, too. Just add at least two whitespaces at the opposite end of your text: Add two spaces on the left to align right, two spaces on the right to align left and two spaces at least at both ends for centered text.
	+
	+	^ Table with alignment ^^^
	+	\| right\| center \|left \|
	+	\|left \| right\| center \|
	+	\| xxxxxxxxxxxx \| xxxxxxxxxxxx \| xxxxxxxxxxxx \|
	+
	+	This is how it looks in the source:
	+
	+	^ Table with alignment ^^^
	+	\| right\| center \|left \|
	+	\|left \| right\| center \|
	+	\| xxxxxxxxxxxx \| xxxxxxxxxxxx \| xxxxxxxxxxxx \|
	+
	+	Note: Vertical alignment is not supported.
	+
	+	===== No Formatting =====
	+
	+	If you need to display text exactly like it is typed (without any formatting), enclose the area either with ''%%<nowiki>%%'' tags or even simpler, with double percent signs ''<nowiki>%%</nowiki>''.
	+
	+	<nowiki>
	+	This is some text which contains addresses like this: http://www.splitbrain.org and formatting, but nothing is done with it.
	+	</nowiki>
	+	The same is true for %%//__this__ text// with a smiley ;-)%%.
	+
	+	<nowiki>
	+	This is some text which contains addresses like this: http://www.splitbrain.org and formatting, but nothing is done with it.
	+	</nowiki>
	+	The same is true for %%//__this__ text// with a smiley ;-)%%.
	+
	+	===== Code Blocks =====
	+
	+	You can include code blocks into your documents by either indenting them by at least two spaces (like used for the previous examples) or by using the tags ''%%<code>%%'' or ''%%<file>%%''.
	+
	+	This is text is indented by two spaces.
	+
	+	<code>
	+	This is preformatted code all spaces are preserved: like <-this
	+	</code>
	+
	+	<file>
	+	This is pretty much the same, but you could use it to show that you quoted a file.
	+	</file>
	+
	+	Those blocks were created by this source:
	+
	+	This is text is indented by two spaces.
	+
	+	<code>
	+	This is preformatted code all spaces are preserved: like <-this
	+	</code>
	+
	+	<file>
	+	This is pretty much the same, but you could use it to show that you quoted a file.
	+	</file>
	+
	+	==== Syntax Highlighting ====
	+
	+	[[wiki:DokuWiki]] can highlight sourcecode, which makes it easier to read. It uses the [[http://qbnz.com/highlighter/\|GeSHi]] Generic Syntax Highlighter -- so any language supported by GeSHi is supported. The syntax uses the same code and file blocks described in the previous section, but this time the name of the language syntax to be highlighted is included inside the tag, e.g. ''<nowiki><code java></nowiki>'' or ''<nowiki><file java></nowiki>''.
	+
	+	<code java>
	+	/**
	+	* The HelloWorldApp class implements an application that
	+	* simply displays "Hello World!" to the standard output.
	+	*/
	+	class HelloWorldApp {
	+	public static void main(String[] args) {
	+	System.out.println("Hello World!"); //Display the string.
	+	}
	+	}
	+	</code>
	+
	+	The following language strings are currently recognized: //4cs 6502acme 6502kickass 6502tasm 68000devpac abap actionscript3 actionscript ada aimms algol68 apache applescript apt_sources arm asm asp asymptote autoconf autohotkey autoit avisynth awk bascomavr bash basic4gl batch bf biblatex bibtex blitzbasic bnf boo caddcl cadlisp ceylon cfdg cfm chaiscript chapel cil c_loadrunner clojure c_mac cmake cobol coffeescript c cpp cpp-qt cpp-winapi csharp css cuesheet c_winapi dart dcl dcpu16 dcs delphi diff div dos dot d ecmascript eiffel email epc e erlang euphoria ezt f1 falcon fo fortran freebasic freeswitch fsharp gambas gdb genero genie gettext glsl gml gnuplot go groovy gwbasic haskell haxe hicest hq9plus html html4strict html5 icon idl ini inno intercal io ispfpanel java5 java javascript jcl j jquery julia kixtart klonec klonecpp kotlin latex lb ldif lisp llvm locobasic logtalk lolcode lotusformulas lotusscript lscript lsl2 lua m68k magiksf make mapbasic mathematica matlab mercury metapost mirc mk-61 mmix modula2 modula3 mpasm mxml mysql nagios netrexx newlisp nginx nimrod nsis oberon2 objc objeck ocaml-brief ocaml octave oobas oorexx oracle11 oracle8 oxygene oz parasail parigp pascal pcre perl6 perl per pf phix php-brief php pic16 pike pixelbender pli plsql postgresql postscript povray powerbuilder powershell proftpd progress prolog properties providex purebasic pycon pys60 python qbasic qml q racket rails rbs rebol reg rexx robots roff rpmspec rsplus ruby rust sas sass scala scheme scilab scl sdlbasic smalltalk smarty spark sparql sql sshconfig standardml stonescript swift systemverilog tclegg tcl teraterm texgraph text thinbasic tsql twig typoscript unicon upc urbi uscript vala vbnet vb vbscript vedit verilog vhdl vim visualfoxpro visualprolog whitespace whois winbatch wolfram xbasic xml xojo xorg_conf xpp yaml z80 zxbasic//
	+
	+	There are additional [[doku>syntax_highlighting\|advanced options]] available for syntax highlighting, such as highlighting lines or adding line numbers.
	+
	+	==== Downloadable Code Blocks ====
	+
	+	When you use the ''%%<code>%%'' or ''%%<file>%%'' syntax as above, you might want to make the shown code available for download as well. You can do this by specifying a file name after language code like this:
	+
	+	<code>
	+	<file php myexample.php>
	+	<?php echo "hello world!"; ?>
	+	</file>
	+	</code>
	+
	+	<file php myexample.php>
	+	<?php echo "hello world!"; ?>
	+	</file>
	+
	+	If you don't want any highlighting but want a downloadable file, specify a dash (''-'') as the language code: ''%%<code - myfile.foo>%%''.
	+
	+	===== RSS/ATOM Feed Aggregation =====
	+	[[DokuWiki]] can integrate data from external XML feeds. For parsing the XML feeds, [[http://simplepie.org/\|SimplePie]] is used. All formats understood by SimplePie can be used in DokuWiki as well. You can influence the rendering by multiple additional space separated parameters:
	+
	+	^ Parameter ^ Description ^
	+	\| any number \| will be used as maximum number items to show, defaults to 8 \|
	+	\| reverse \| display the last items in the feed first \|
	+	\| author \| show item authors names \|
	+	\| date \| show item dates \|
	+	\| description\| show the item description. All HTML tags will be stripped \|
	+	\| nosort \| do not sort the items in the feed \|
	+	\| //n//[dhm] \| refresh period, where d=days, h=hours, m=minutes. (e.g. 12h = 12 hours). \|
	+
	+	The refresh period defaults to 4 hours. Any value below 10 minutes will be treated as 10 minutes. [[wiki:DokuWiki]] will generally try to supply a cached version of a page, obviously this is inappropriate when the page contains dynamic external content. The parameter tells [[wiki:DokuWiki]] to re-render the page if it is more than //refresh period// since the page was last rendered.
	+
	+	By default the feed will be sorted by date, newest items first. You can sort it by oldest first using the ''reverse'' parameter, or display the feed as is with ''nosort''.
	+
	+	Example:
	+
	+	{{rss>http://slashdot.org/index.rss 5 author date 1h }}
	+
	+	{{rss>http://slashdot.org/index.rss 5 author date 1h }}
	+
	+
	+	===== Control Macros =====
	+
	+	Some syntax influences how DokuWiki renders a page without creating any output it self. The following control macros are availble:
	+
	+	^ Macro ^ Description \|
	+	\| %%~~NOTOC~~%% \| If this macro is found on the page, no table of contents will be created \|
	+	\| %%~~NOCACHE~~%% \| DokuWiki caches all output by default. Sometimes this might not be wanted (eg. when the %%<php>%% syntax above is used), adding this macro will force DokuWiki to rerender a page on every call \|
	+
	+	===== Syntax Plugins =====
	+
	+	DokuWiki's syntax can be extended by [[doku>plugins\|Plugins]]. How the installed plugins are used is described on their appropriate description pages. The following syntax plugins are available in this particular DokuWiki installation:
	+
	+	~~INFO:syntaxplugins~~

synchrotron_applications

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

2015/02/10 17:36		current
Line 9:		Line 9:
	* 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.

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 10:01		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> faq file_format ptu open_source analysis}}	+	{{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:

novaflim_license

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T15:50:52+00:00

2025/02/14 16:40		current
Line 1:		Line 1:
	====== NovaFLIM Licenses ======		====== NovaFLIM Licenses ======
		+	{{tag> faq software NovAFLIM license}}
		+

	For NovaFLIM, we use a software license instead of a hardware dongle to make license management more efficient. Compared to a hardware dongle, this approach eliminates the time and costs associated with handling and shipping dongles, as well as the risk of losing or removing them.		For NovaFLIM, we use a software license instead of a hardware dongle to make license management more efficient. Compared to a hardware dongle, this approach eliminates the time and costs associated with handling and shipping dongles, as well as the risk of losing or removing them.

why_do_i_not_get_the_same_count_rates_on_both_spad_detectors

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T15:47:18+00:00

2025/02/14 16:45		current
Line 1:		Line 1:
	====== 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? ======
-	{{tag> detectors faq SPAD}}	+	{{tag> detectors faq SPAD MT200}}

	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).

bootstrap

Anonymous (anonymous@undisclosed.example.com) — 2014-01-21T16:41:23+00:00

2014/01/21 17:41		current
Line 4:		Line 4:

	Specifically, the bootstrap analysis takes a subset of N randomly chosen points out of the experimental data set, which itself consists of N data points. Since the points are chosen randomly, some of the points will be selected more than once; others will not be selected at all. Thus the 'simulated' data set is not identical to the original one, while the statistical properties of the original data set are maintained (since the number of data points remains the same).		Specifically, the bootstrap analysis takes a subset of N randomly chosen points out of the experimental data set, which itself consists of N data points. Since the points are chosen randomly, some of the points will be selected more than once; others will not be selected at all. Thus the 'simulated' data set is not identical to the original one, while the statistical properties of the original data set are maintained (since the number of data points remains the same).
-
-	At PQ the bootstrap method is exclusively used in [[FluoFit]]. For ensemble fitting as in [[FLIM]] it is -alas!- way too slow.
-

	===== Advantages and disadvantages =====		===== Advantages and disadvantages =====

convolution

Anonymous (anonymous@undisclosed.example.com) — 2013-08-06T14:39:35+00:00

2013/08/06 16:38		current
Line 1:		Line 1:
	====== Convolution ======		====== Convolution ======

-	The convolution $C$ of a function $F(t)$ with a function $G(t)$ (of a parameter $t$) is defined as	+	The convolution $C$ of a function $F$ with a function $G$ (of a parameter $t$) is defined as

	$C(t)=\int_{-\infty}^t F(t-t')~G(t')~dt'$		$C(t)=\int_{-\infty}^t F(t-t')~G(t')~dt'$

fluorescence_lifetime

Anonymous (anonymous@undisclosed.example.com) — 2014-04-09T21:12:33+00:00

2026/04/23 19:16	current
Line 1:	Line 1:
	+	====== Fluorescence Lifetime ======

	+	[[wp>Fluorescence#Lifetime]]

pie

Anonymous (anonymous@undisclosed.example.com) — 2013-08-06T14:33:18+00:00

2013/07/31 10:40		current
Line 1:		Line 1:
	====== Pulsed Interleaved Excitation (PIE) ======		====== Pulsed Interleaved Excitation (PIE) ======

-	PIE stands for Pulsed Interleaved Excitation: a technique of synchronising several pulsed lasers (or other suitable excitation sources). The laser pulses are separated on a ns time scale to allow simultaneous recording of the time response of a sample molecule due to each individual laser.	+	PIE stands for Pulsed Interleaved Excitation: a technique of synchronising several pulsed lasers (or other suitable excitation sources). The laser pulses are separated on a ns time scale to allow simultaneous recording of the time response of a sample molecule due to each individual laser.

reconvolution

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

2014/04/09 22:38		current
Line 3:		Line 3:
	Reconvolution is used in time domain data analysis for removal of broadening effects due to instrumental resolution. In contrast to [[deconvolution]], data processing using reconvolution techniques does not aim at retrieving the raw data as seen by an ideal instrument, but does usually compare artificial decays (simulated on the base of a model) convolved with the measured [[IRF]].		Reconvolution is used in time domain data analysis for removal of broadening effects due to instrumental resolution. In contrast to [[deconvolution]], data processing using reconvolution techniques does not aim at retrieving the raw data as seen by an ideal instrument, but does usually compare artificial decays (simulated on the base of a model) convolved with the measured [[IRF]].

-	Reconvolution is used in PicoQuant software packages (mainly [[products:FluoFit]] and [[products:SymPhoTime]]) for compensating IRF effects.	+	Reconvolution is used in PicoQuant software packages (mainly [[software:FluoFit]] and [[software:SymPhoTime]]) for compensating IRF effects.

2ffcs

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

2014/04/09 22:02		current
Line 7:		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 =====

flim_measurement_using_a_nikon_a1_with_a_flim_and_fcs_upgrade

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

2015/06/16 13:47		current
Line 15:		Line 15:

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

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

reconvolution_fit

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

2022/08/18 13:30		current
Line 14:		Line 14:


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

picoharp_300

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

2015/02/10 17:55		current
Line 7:		Line 7:

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

faq

Anonymous (anonymous@undisclosed.example.com) — 2025-03-14T09:40:11+00:00

2025/03/14 10:34		current
Line 19:		Line 19:

	{{topic> faq -draft &nodate&nouser}}		{{topic> faq -draft &nodate&nouser}}
-	[[FAQ:old dongles drivers for FluoFit SymPhoTime 32:old dongles drivers for FluoFit SymPhoTime 32\|Using old dongles for FluoFit and older versions of SymPhoTime (SymPhoTime 32) with Windows 10 or 11]]

imprint

Anonymous (anonymous@undisclosed.example.com) — 2018-05-23T08:30:11+00:00

2018/05/23 10:29		current
Line 11:		Line 11:
	https://www.picoquant.com\\		https://www.picoquant.com\\

-	Vertretungsberechtiger Geschäftsführer/General Manager: Rainer Erdmann	+	Vertretungsberechtiger Geschäftsführer/General Manager: Rainer Erdmann\\
-	HRB 60901 Berlin-Charlottenburg	+	HRB 60901 Berlin-Charlottenburg\\
-	VAT ID: DE 812140373	+	VAT ID: DE 812140373\\
-	WEEE-Reg.-Nr. DE 96457402	+	WEEE-Reg.-Nr. DE 96457402\\
-	Tax-ID.: 37/098/45817	+	Tax-ID.: 37/098/45817\\

lsm710

Anonymous (anonymous@undisclosed.example.com) — 2016-07-21T13:31:38+00:00

2016/07/20 14:05		current
Line 2:		Line 2:


-	====== FLIM Measurement Using a Zeiss LSM710 with a FLIM and FCS Upgrade ======	+	====== FLIM Measurement Using a Zeiss LSM710/LSM780/LSM880 with a FLIM and FCS Upgrade ======

	This tutorial shows the recording of FLIM images using an LSM upgrade kit, in this case a Zeiss LSM710.		This tutorial shows the recording of FLIM images using an LSM upgrade kit, in this case a Zeiss LSM710.
Line 11:		Line 11:
	* to monitor concentration changes using a reporter dye		* to monitor concentration changes using a reporter dye

-	* To monitor interactions between two molecules via FRET.	+	* To monitor interactions between two molecules via FRET. \\
		+
		+
		+	\\
		+	{{ youtube>QXGqA2IsivM?large }} \\

	The data acquisition is analogous also for upgrades on an LSM780 or LSM880, and the general principles of the data acquisition are analogous in all LSM upgrade kits.		The data acquisition is analogous also for upgrades on an LSM780 or LSM880, and the general principles of the data acquisition are analogous in all LSM upgrade kits.
		+	\\ \\
		+	\\
		+	{{ youtube>vofLPLVmq-Q?large }} \\

-	{{ youtube>QXGqA2IsivM?large }}

novaflim_calculated_irf

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

2025/02/18 14:49		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.

asymptotic_standard_errors

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:33:41+00:00

2014/04/09 22:41		current
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	The asymptotic standard errors (ASE) are used for analyzing fitting parameter error intervals. For illustration let's start at the best fit parameter set, which can be regarded as a single point in the parameter space. Now we remove the parameter for which we want to calculate the error intervals from this location, that is, we take a single step parallel to its parameter axis. We then calculate the reduced X<sup>2</sup> for this new paramter set. By iterating this procedure we get X<sup>2</sup> as a function of the parameter of interest. The intersection points of this function with a given X<sup>2</sup> confidence limit define the boundaries of the confidence interval of the parameter.		The asymptotic standard errors (ASE) are used for analyzing fitting parameter error intervals. For illustration let's start at the best fit parameter set, which can be regarded as a single point in the parameter space. Now we remove the parameter for which we want to calculate the error intervals from this location, that is, we take a single step parallel to its parameter axis. We then calculate the reduced X<sup>2</sup> for this new paramter set. By iterating this procedure we get X<sup>2</sup> as a function of the parameter of interest. The intersection points of this function with a given X<sup>2</sup> confidence limit define the boundaries of the confidence interval of the parameter.

-	At PQ the asymptotic standard errors are supported by [[products:FluoFit]] and the [[products:SymPhoTime]] software.	+	At PQ the asymptotic standard errors are supported by [[software:FluoFit]] and the [[software:SymPhoTime]] software.

bdf

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:44:39+00:00

2015/02/10 17:44		current
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	====== BDF ======		====== BDF ======

-	Binary Data File: files calculated by the [[software:SymPhoTime32]] software analyzing a TTTR file.	+	Binary Data File: files calculated by the [[software:SymPhoTime 32]] software analyzing a TTTR file.

fastlt

Anonymous (anonymous@undisclosed.example.com) — 2013-08-06T13:52:06+00:00

2013/08/06 15:51		current
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-	#redirect [[Fast Lifetime]]	+	#redirect [[glossary:Fast Lifetime]]

fcs

Anonymous (anonymous@undisclosed.example.com) — 2015-01-22T14:55:15+00:00

2015/01/22 15:54		current
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	====== Fluorescence Correlation Spectroscopy (FCS) ======		====== Fluorescence Correlation Spectroscopy (FCS) ======
		+	Fluorescence correlation spectroscopy (FCS) is a correlation analysis of fluctuation of the fluorescence intensity. The analysis provides parameters of the physics under the fluctuations. One of the interesting applications of this is an analysis of the concentration fluctuations of fluorescent particles (molecules) in solution. In this application, the fluorescence emitted from a very tiny space in solution containing a small number of fluorescent particles (molecules) is observed. The fluorescence intensity is fluctuating due to Brownian motion of the particles. In other words, the number of the particles in the sub-space defined by the optical system is randomly changing around the average number. The analysis gives the average number of fluorescent particles and average diffusion time, when the particle is passing through the space. Eventually, both the concentration and size of the particle (molecule) are determined. Both parameters are important in biochemical research, biophysics, and chemistry.(([[http://en.wikipedia.org/wiki/Fluorescence_correlation_spectroscopy]]))
		+

	see [[general:fluorescence_correlation_spectroscopy-_a_short_introduction\|Introduction to Fluorescence Correlation Spectroscopy]].		see [[general:fluorescence_correlation_spectroscopy-_a_short_introduction\|Introduction to Fluorescence Correlation Spectroscopy]].

irf

Anonymous (anonymous@undisclosed.example.com) — 2014-02-21T13:28:43+00:00

2014/02/21 14:28		current
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	IRF stands for Instrument Response Function. The IRF is the best approximation to a temporally infinitely short process possible to measure with a given instrument. A synonyme sometimes used is 'lamp function', which stresses the fact that the temporal profile of the excitation is the most intuitive contribution to the IRF. Of course, the temporal resolution of all components of the instrument contribute to both shape and width of the IRF. The observed decay in a time domain measurement is a [[convolution]] of the 'real' decay with the IRF.		IRF stands for Instrument Response Function. The IRF is the best approximation to a temporally infinitely short process possible to measure with a given instrument. A synonyme sometimes used is 'lamp function', which stresses the fact that the temporal profile of the excitation is the most intuitive contribution to the IRF. Of course, the temporal resolution of all components of the instrument contribute to both shape and width of the IRF. The observed decay in a time domain measurement is a [[convolution]] of the 'real' decay with the IRF.

-	see also [[howto:how_to_measure_the_instrument_response_function_irf\|[[howto:How To Measure the IRF]]]]	+	see also [[howto:how_to_measure_the_instrument_response_function_irf\|How To Measure the IRF]]

least_squares

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

2014/04/09 22:42		current
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	====== 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

mcp

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T13:27:59+00:00

2013/08/05 13:45		current
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	====== MCP ======		====== MCP ======

-	Microchannel Plate PMT (MCP)	+	Microchannel Plate [[PMT]] (MCP)

-	A microchannel plate PMT is a sensitive photon detector. It consists of an array of glass capillaries (10-25 μm inner diameter) that are coated on the inside with a electron-emissive material. The capillaries are biased at a high voltage. Like in the PMT, an electron that strikes the inside wall of one of the capillaries creates an avalanche of secondary electrons. This cascading effect creates a gain of 103 to 106 and produces a current pulse at the output. The timing jitter of MCPs is sufficiently small to perform time-resolved photon counting on a sub-nanosecond- scale, usually outperforming PMTs. Good but also expensive MCPs can achieve timing uncertainties as low as 20ps. Microchannel plates are also used as an intensifier for low-intensity light detection with array detectors.	+	A microchannel plate [[PMT]] is a sensitive photon detector. It consists of an array of glass capillaries (10-25 μm inner diameter) that are coated on the inside with a electron-emissive material. The capillaries are biased at a high voltage. Like in the PMT, an electron that strikes the inside wall of one of the capillaries creates an avalanche of secondary electrons. This cascading effect creates a gain of 103 to 106 and produces a current pulse at the output. The timing jitter of MCPs is sufficiently small to perform time-resolved photon counting on a sub-nanosecond- scale, usually outperforming PMTs. Good but also expensive MCPs can achieve timing uncertainties as low as 20ps. Microchannel plates are also used as an intensifier for low-intensity light detection with array detectors.

pre-histogrammed_image

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

2018/01/24 15:40		current
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	====== 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 \|

residuals

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

2014/04/09 22:45		current
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	$$w_i=\sqrt{D_i^{exp}}$$		$$w_i=\sqrt{D_i^{exp}}$$

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

spad

Anonymous (anonymous@undisclosed.example.com) — 2017-05-17T08:37:15+00:00

2013/08/06 16:53		current
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	SPAD means Single Photon counting Avalanche photo-Diode. It stands commonly for a complete module including an Avalanche Photodiode, working temporary above the breakdown voltage in reverse bias. One photon may trigger the avalanche process and generates a time-corresponding electrical pulse. The avalanche must be "quenched" in order to prevent destructive current flow. This is achieved by passive or active quenching circuits. Passive quenching requires basically just a resistor but is slow and causes longer deadtimes. Active quenching is standard for all modules used by PicoQuant.		SPAD means Single Photon counting Avalanche photo-Diode. It stands commonly for a complete module including an Avalanche Photodiode, working temporary above the breakdown voltage in reverse bias. One photon may trigger the avalanche process and generates a time-corresponding electrical pulse. The avalanche must be "quenched" in order to prevent destructive current flow. This is achieved by passive or active quenching circuits. Passive quenching requires basically just a resistor but is slow and causes longer deadtimes. Active quenching is standard for all modules used by PicoQuant.

-	/*
-	[[Howto:How to unmount a SPAD from the MT200]]
-	*/

flim-fret_measurement_using_an_olympus_fv1200_with_a_flim_and_fcs_upgrade

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

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

Line 20:		Line 20:
	{{topic>howto +IRF &nodate&nouser}}		{{topic>howto +IRF &nodate&nouser}}
	{{topic>howto +FLIM +FRET +analysis &nodate&nouser}}		{{topic>howto +FLIM +FRET +analysis &nodate&nouser}}
-	{{topic>howto +FLIM +Nikon}}

recording_a_fluorescence_lifetime_image_flim_stack_with_a_lsm_upgrade_kit_on_a_nikon_a1

Anonymous (anonymous@undisclosed.example.com) — 2015-06-30T13:04:26+00:00

2015/02/25 14:56		current
Line 4:		Line 4:


-	<WRAP center important 80%>	+
-	DRAFT	+	This tutorial shows the integration with NIS version <4.3.
-	</WRAP>	+

tcspc_electronics

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

2026/04/23 19:16	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]]

	+

flimfit

Anonymous (anonymous@undisclosed.example.com) — 2014-05-23T09:04:11+00:00

2014/05/23 11:02		current
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-	{{tag> software analysis FLIM }}	+	{{tag> software analysis FLIM open_source}}

	====== FLIMfit ======		====== FLIMfit ======

supported_mt200_pc_configuration_for_symphotime_32

Anonymous (anonymous@undisclosed.example.com) — 2014-05-23T07:36:46+00:00

2026/04/23 19:16		current
Line 1:		Line 1:
		+	This page is redirected to [[:software:supported_mt200_pc_configuration_for_symphotime_32]].

supported_mt200_pc_configuration_for_symphotime_64

Anonymous (anonymous@undisclosed.example.com) — 2014-05-23T07:33:10+00:00

2026/04/23 19:16		current
Line 1:		Line 1:
		+	This page is redirected to [[:software:supported_mt200_pc_configuration_for_symphotime_64]].

2019/03/06 13:44		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:
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	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 $k_{AB}$ 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.		//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]].