Time-Resolved Fluorescence Wiki

calculate_ratiometric_single_pair_fret_distributions_using_the_pie-fret_script

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

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

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.\\

	\\		\\

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 ======

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 ======

applications

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

2023/02/23 09:12		current
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	[[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]]

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
Line 85:		Line 85:

	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".

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.

using_the_antibunching_script

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

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

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

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

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 ======

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.

some_origins_of_multiexponetial_decays_for_single_dyes

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

diamond_nv_centers

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

2018/04/19 14:11		current
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	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 ====

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.\\

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}}	+

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
Line 1:		Line 1:
-	{{tag>howto FLIM multi-frame imaging analysis dynamics tutorial LSM_upgrade SPT SymPhoTime}}	+	{{tag>howto FLIM multi-frame imaging analysis dynamics tutorial LSM_upgrade SymPhoTime}}

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

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 ======

symphotime_tips_and_tricks

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

2020/04/03 16:54		current
Line 1:		Line 1:

-	~~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 =====

support_plane_analysis

Anonymous (anonymous@undisclosed.example.com) — 2014-01-21T16:46:58+00:00

2014/01/21 17:46		current
Line 13:		Line 13:
	===== References =====		===== References =====

-	Lakowicz JR (1999)// Principles of Fluorescence Spectroscopy//, 2nd edn. Kluver Academic/Plenum Publishers, New York (available in room C202)*	+	*Lakowicz JR (1999)// Principles of Fluorescence Spectroscopy//, 2nd edn. Kluver Academic/Plenum Publishers, New York

antibunching_measurements

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

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

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

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

calculate_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 ======

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.

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
Line 50:		Line 50:
	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_6.png?600 }}		{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_6.png?600 }}

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

	* Click: "Initial Fit" (marked in orange).		* Click: "Initial Fit" (marked in orange).
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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.

roi_fitting_using_the_flim_script

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

2020/05/13 11:42		current
Line 9:		Line 9:


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

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

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

asymptotic_standard_errors

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

2014/04/09 22:41		current
Line 5:		Line 5:
	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.

pile-up_effect

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

2015/06/03 17:09		current
Line 1:		Line 1:
-	{{tag>pile-up TCSPC pile-up dead_time}}	+	{{tag> TCSPC pile-up dead_time}}
	~~TOC~~		~~TOC~~

intensity_time_trace_analysis

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

2014/06/19 10:12		current
Line 1:		Line 1:
	{{tag> tutorial howto time-trace analysis SymPhoTime}}		{{tag> tutorial howto time-trace analysis SymPhoTime}}

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

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

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 \|}}
	\\		\\

separation_of_2_species_with

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

2014/06/18 12:57		current
Line 1:		Line 1:
	{{tag> howto tutorial FCS FLCS analysis SymPhoTime}}		{{tag> howto tutorial FCS FLCS analysis SymPhoTime}}

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

	====== Separation of 2 Species with Different Lifetimes Using 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]].
-

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
Line 3:		Line 3:
	~~NOTOC~~		~~NOTOC~~

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

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

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

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]]

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>

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) ======

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.

hybrid_pmt

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T15:00:37+00:00

2015/11/03 15:59		current
Line 3:		Line 3:
	"The hybrid photodetector's structure is similar yet different from a conventional PMT. Like PMTs, the HPD is a vacuum tube with a photocathode that detects light, an electron multiplier that multiplies electrons, and an output terminal that outputs an electrical signal. But unlike PMTs which use multiple dynodes as electron multipliers, the HPD uses a silicon avalanche diode (AD) instead"((continue reading on the [[http://www.hamamatsu.com/us/en/community/optical_sensors/tutorials/what_is_hpd/index.html\|Hamamatsu]] website.))		"The hybrid photodetector's structure is similar yet different from a conventional PMT. Like PMTs, the HPD is a vacuum tube with a photocathode that detects light, an electron multiplier that multiplies electrons, and an output terminal that outputs an electrical signal. But unlike PMTs which use multiple dynodes as electron multipliers, the HPD uses a silicon avalanche diode (AD) instead"((continue reading on the [[http://www.hamamatsu.com/us/en/community/optical_sensors/tutorials/what_is_hpd/index.html\|Hamamatsu]] website.))

-	PicoQuant integrates various Hybrid PMT tubes into a convenient assembly called [[https://www.picoquant.com/products/category/photon-counting-detectors/pma-hybrid-series-hybrid-photomultiplier-detector-assembly\|PMA Hybrid]].	+	PicoQuant integrates Hybrid PMT tubes into a convenient assembly called [[https://www.picoquant.com/products/category/photon-counting-detectors/pma-hybrid-series-hybrid-photomultiplier-detector-assembly\|PMA Hybrid]].

	The PMA Hybrid is a compact single photon sensitive detector based on a fast hybrid photomultiplier tube with peltier cooler to reduce the dark count rate. The detector includes a high voltage power supply and pre-amplifier with overload protection and emergency shut down procedure if the detector count rate reaches a critical limit.		The PMA Hybrid is a compact single photon sensitive detector based on a fast hybrid photomultiplier tube with peltier cooler to reduce the dark count rate. The detector includes a high voltage power supply and pre-amplifier with overload protection and emergency shut down procedure if the detector count rate reaches a critical limit.

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.

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.

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 =====

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.

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).\\

data_file_import

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

2022/08/04 14:53		current
Line 26:		Line 26:
	\\		\\
	After expanding the data container:		After expanding the data container:
-	{{ :howto:et2_workspace2.png?340 \|}}	+	{{ :howto:et2_workspace2.png \|}}

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

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 }}

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}}

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 ====

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
Line 2:		Line 2:
	~~NOTOC~~		~~NOTOC~~

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

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 =====

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]].

tcspc_electronics

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

2026/04/23 19:26	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]]

	+

syntax

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

2026/04/23 19:26	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 ^^^
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	+
	+	This is how it looks in the source:
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	+	^ Table with alignment ^^^
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	+	~~INFO:syntaxplugins~~

phasor_plot_structure_separation

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

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

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

	Introduction		Introduction

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.
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-	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 $\tau_A$.
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	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 $t=0$.
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	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]].