Time-Resolved Fluorescence Wiki

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 ======

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 ======

video_tutorials

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

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

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

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

how_to_measure_the_instrument_response_function_irf

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

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

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

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

using_the_flcs_script_for_spectral_crosstalk_removal_via_flccs

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

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

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

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 ====

some_origins_of_multiexponetial_decays_for_single_dyes

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

measurement_hardware_instrumentation

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

2018/06/25 09:17		current
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	[[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]]

fcs

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

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

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

calculate_ratiometric_single_pair_fret_distributions

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

2014/06/18 17:56		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 SPAD or Hybrid PMA are necessary to successfully detect single molecule fluorescence.\\	+	To record such traces, usually single molecule sensitive detectors as [[glossary:SPAD]] or [[glossary:Hybrid PMT]]s are necessary to successfully detect single molecule fluorescence.\\

	\\		\\

calculate_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.

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.

basics

Anonymous (anonymous@undisclosed.example.com) — 2024-10-11T08:23:47+00:00

2021/09/23 14:59		current
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	{{tag> basics introduction FCS fluorescence microscopy objective resolution NA tutorials}}		{{tag> basics introduction FCS fluorescence microscopy objective resolution NA tutorials}}
-	===== Basics =====	+	====== Basics ======

	* [[video_tutorials\|Fluorescence and Fluorescence Microscopy by Nico Stuurman]]		* [[video_tutorials\|Fluorescence and Fluorescence Microscopy by Nico Stuurman]]

fluorescence_lifetime_measurements_using_the_fluotime_300

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

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

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

microtime

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

2015/11/03 15:23		current
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	* 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

using_the_anisotropy_image_script

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

2018/04/12 12:26		current
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	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 ====

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]]


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	[[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]]

start

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

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

antibunching_measurements

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

2025/04/09 16:00		current
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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
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	~~NOTOC~~		~~NOTOC~~

-	====== Calculate Multiple FCCS Traces with the Grouped FCS Script ======	+	====== Calculate FCCS Traces with the Grouped FCS Script ======

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

flim_fret_calculation_for_multi_exponential_donors

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

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

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

separation_of_2_species_with

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

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

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

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

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

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

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.
-

visualizing_dynamics_with_the_multi_frame_flim_analysis

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

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

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

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

lifetime_component_decomposition_phasor_plot_analysis

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

2025/06/16 09:17		current
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	* 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

pile-up_effect

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

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

flim-fret_calculation_for_single_exponential_donors

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

2020/05/06 11:19		current
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	Response: In the image, only pixels with higher photon counts are highlighted. Adapt the intensity maximum to see the highlighted pixels more clearly.		Response: In the image, only pixels with higher photon counts are highlighted. Adapt the intensity maximum to see the highlighted pixels more clearly.
-	{{ :howto:flim-fret-1expd_roi_from_thresh_2_v2.png?400 \|}}	+	{{ :howto:flim-fret-1expd_roi_from_thresh_2_v3.png?400 \|}}

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

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.\\

visualizing_dynamics_using_the_multiframe-flim_script

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

2021/10/19 21:59		current
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-	{{tag>howto FLIM multi-frame imaging analysis dynamics tutorial LSM_upgrade SPT SymPhoTime}}	+	{{tag>howto FLIM multi-frame imaging analysis dynamics tutorial LSM_upgrade SymPhoTime}}

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

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

how_to_reinstall_symphotime

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

2025/04/09 12:55		current
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	Updating from Windows 10 to Windows 11 should work smoothly, and all software should stay as it is. However it is a good idea to back-up the files mentioned below.		Updating from Windows 10 to Windows 11 should work smoothly, and all software should stay as it is. However it is a good idea to back-up the files mentioned below.
		+
	Please note: The Framegrabber MOU camera is not compatible with Windows 11 Windows 11 works with the newer USB MOU camera.		Please note: The Framegrabber MOU camera is not compatible with Windows 11 Windows 11 works with the newer USB MOU camera.
	==== Getting everything from the old computer ====		==== Getting everything from the old computer ====

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
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-	{{tag>TPE}}
	====== Advantages and disadvantages of two photon excitation (TPE) ======		====== Advantages and disadvantages of two photon excitation (TPE) ======

flim

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

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

fluorescence_lifetime

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

2026/04/23 21:31	current
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	+	====== Fluorescence Lifetime ======

	+	[[wp>Fluorescence#Lifetime]]

flim_measurement_using_a_nikon_a1_with_a_flim_and_fcs_upgrade

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

2015/06/16 13:47		current
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	The general principles of the data acquisition are analogous in all LSM upgrade kits.		The general principles of the data acquisition are analogous in all LSM upgrade kits.
-	The movie was recorded in 02/2014; the recording procedure may be somewhat different in older or newer	+	The movie was recorded in 02/2014 and shows the integration with Nikon A1 system with a NIS version lower than 4.3; the recording procedure may be somewhat different in higher or very old versions.
-	models.	+

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

intensity_time_trace_analysis

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

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

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

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

lifetime_fitting_using_the_flim_analysis

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

2014/06/18 14:19		current
Line 3:		Line 3:
	~~NOTOC~~		~~NOTOC~~

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

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}}	+

roi_fitting_using_the_flim_script

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

2020/05/13 11:42		current
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-	This tutorial shows step-by-step, how the [[glossary:flim\|FLIM]] script of [[products:SymPhoTime64\|SymPhoTime 64]] can be used to fit several regions of interest (ROIs), and how to extract and interpret the results. In detail, the FLIM script is started using a daisy pollen image from the "Samples" – workspace, then three regions of interest are defined which are finally fitted.	+	This tutorial shows step-by-step, how the [[glossary:flim\|FLIM]] script of [[products:SymPhoTime64\|SymPhoTime 64]] can be used to fit several regions of interest (ROIs), and how to extract and interpret the results. In details, the FLIM script is started using a daisy pollen image from the "Samples" – workspace, then three regions of interest are defined which are finally fitted.

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

start

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

2024/12/19 16:57		current
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	{{topic>howto -FT300 -sample -analysis -draft &nodate&nouser}}		{{topic>howto -FT300 -sample -analysis -draft &nodate&nouser}}

-	===== NovaFLIM / Analysis =====
-
-	{{topic>howto +tutorial +novaflim -draft &nodate&nouser}}

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

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.

fluofit

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:07:12+00:00

2015/11/03 15:07		current
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	====== FluoFit ======		====== FluoFit ======
-	The FluoFit software package is a powerful global analysis software for fluorescence decay and anisotropy measurements. Tail fitting as well as a numerical reconvolution algorithm to account for the finite Instrument Response Function ([[IRF]]) can be applied. The FluoFit software is available in two versions (Basic and Professional) with different functionalities to meet individual needs.	+	The FluoFit software package is a powerful global analysis software for fluorescence decay and anisotropy measurements. Tail fitting as well as a numerical reconvolution algorithm to account for the finite Instrument Response Function ([[glossary:IRF]]) can be applied. The FluoFit software is available in two versions (Basic and Professional) with different functionalities to meet individual needs.

	see [[https://www.picoquant.com/products/category/software/fluofit-global-fluorescence-decay-data-analysis-software]]		see [[https://www.picoquant.com/products/category/software/fluofit-global-fluorescence-decay-data-analysis-software]]

pycorrfit

Anonymous (anonymous@undisclosed.example.com) — 2016-03-07T11:00:06+00:00

2015/11/03 14:59		current
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	"[...] PyCorrFit is a general-purpose FCS evaluation software that, amongst other formats, supports the established Zeiss ConfoCor3 ~.fcs file format. PyCorrFit comes with several built-in model functions, covering a wide range of applications in standard confocal FCS. In addition, it contains equations dealing with different excitation geometries like total internal reflection (TIR)."		"[...] PyCorrFit is a general-purpose FCS evaluation software that, amongst other formats, supports the established Zeiss ConfoCor3 ~.fcs file format. PyCorrFit comes with several built-in model functions, covering a wide range of applications in standard confocal FCS. In addition, it contains equations dealing with different excitation geometries like total internal reflection (TIR)."

-	[[http://paulmueller.github.io/PyCorrFit/]]	+	[[http://pycorrfit.craban.de/]]

	GitHub:[[https://github.com/paulmueller/PyCorrFit]]		GitHub:[[https://github.com/paulmueller/PyCorrFit]]

lsm710

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

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


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

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

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

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

-	{{ youtube>QXGqA2IsivM?large }}

community_news

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

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

convolution

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

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

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

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

mcs

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

2014/01/21 17:38		current
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	====== 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.

support_plane_analysis

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

2014/01/21 17:46		current
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	===== 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

t2-mode

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

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

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


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

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

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

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

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

t3-mode

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

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

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

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
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	* 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).\\

flim-fret_measurement_using_an_olympus_fv1200_with_a_flim_and_fcs_upgrade

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

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

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

mt200fcs

Anonymous (anonymous@undisclosed.example.com) — 2023-02-17T12:43:50+00:00

2021/06/25 11:07		current
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	======MT200 FCS Measurement ======		======MT200 FCS Measurement ======
-	{{tag>howto mt200 video tutorial}}	+	{{tag>howto MicroTime200 video tutorial}}

	fluorescence correlation spectroscopy (FCS) is a quantitative measurement of the resolution and sensitivity of confocal microscopes. In this tutorial we follow molecular brightness, the count rate per molecule per excitation power, as a well suited parameter for comparison of instrument performance and quality of adjustment. The higher the better: if the system is not optimally aligned, the excitation and/or the detection will be less efficient and the molecular brightness will drop.		fluorescence correlation spectroscopy (FCS) is a quantitative measurement of the resolution and sensitivity of confocal microscopes. In this tutorial we follow molecular brightness, the count rate per molecule per excitation power, as a well suited parameter for comparison of instrument performance and quality of adjustment. The higher the better: if the system is not optimally aligned, the excitation and/or the detection will be less efficient and the molecular brightness will drop.

pattern_matching

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

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

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

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

performing_an_fcs_measurement_with_an_olympus_fv1200_upgrade_kit

Anonymous (anonymous@undisclosed.example.com) — 2015-02-25T13:55:57+00:00

2015/02/06 12:58		current
Line 1:		Line 1:
	{{tag> howto FCS acquisition video FV1200 Olympus LSM_upgrade}}		{{tag> howto FCS acquisition video FV1200 Olympus LSM_upgrade}}
-	====== Performing an FCS measurement with a Olympus FV1200 upgrade kit======	+	====== Performing a FCS measurement with an Olympus FV1200 Upgrade Kit======

phasor_analysis

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

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

	~~TOC~~		~~TOC~~

recording_a_fluorescence_lifetime_image_flim_stack_with_a_lsm_upgrade_kit_on_a_nikon_a1

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

2015/02/25 14:56		current
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-	<WRAP center important 80%>	+
-	DRAFT	+	This tutorial shows the integration with NIS version <4.3.
-	</WRAP>	+

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 =====

flimfit

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

2014/05/23 11:02		current
Line 1:		Line 1:
-	{{tag> software analysis FLIM }}	+	{{tag> software analysis FLIM open_source}}

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

symphotime64

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

2014/05/23 09:34		current
Line 5:		Line 5:


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

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

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

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:
Line 34:		Line 34:
	Scheme 2		Scheme 2

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

	//Case A) The constant kAB is too slow with respect to $\tau_A$ and $\tau_B$.// In this case the compound A would decay to the ground-sate before the excited-state reaction could take place. The decay measured would be single exponential and coincident with $\tau_A$.		//Case A) The constant kAB is too slow with respect to $\tau_A$ and $\tau_B$.// In this case the compound A would decay to the ground-sate before the excited-state reaction could take place. The decay measured would be single exponential and coincident with $\tau_A$.
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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 $t=0$.
Line 72:		Line 72:
	Note from $A(t)$ that, even if $B$ would not be fluorescent, the decay of $A$ will still be biexponential!		Note from $A(t)$ that, even if $B$ would not be fluorescent, the decay of $A$ will still be biexponential!

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

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

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


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


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

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

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

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