Time-Resolved Fluorescence Wiki

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 ======

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>

old_dongles_drivers_for_fluofit_symphotime_32

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

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

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

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

supported_mt200_pc_configuration_for_symphotime_32

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

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

tcspc_external_markers

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

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

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.

how_can_i_analyze_my_data_using_non-picoquant_software

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

2025/01/31 10:01		current
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	====== How can I analyze my data using non-PicoQuant software? ======		====== How can I analyze my data using non-PicoQuant software? ======

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

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

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.

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 ======

hydraharp_400

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

2015/09/23 13:43		current
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	The HydraHarp 400 is a high-end, easy to use, plug and play Time Interval Analyzer (TIA) and Time-Correlated Single Photon Counting (TCSPC) system with scalability for multiple channels. It is connected to a PC through a USB 3.0 high speed interface.		The HydraHarp 400 is a high-end, easy to use, plug and play Time Interval Analyzer (TIA) and Time-Correlated Single Photon Counting (TCSPC) system with scalability for multiple channels. It is connected to a PC through a USB 3.0 high speed interface.

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

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

picoharp_300

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

2015/02/10 17:55		current
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	===== Software =====		===== Software =====
-	Like all PicoQuant TCSPC devices the PicoHarp 300 comes with dedicated software for acquisition of [[glossary:T3-data]] and [[glossary:T2-data]].	+	Like all PicoQuant TCSPC devices the PicoHarp 300 comes with dedicated software for acquisition of [[glossary:T3-mode]] and [[glossary:T2-mode]] data.

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

marquardt-levenberg

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

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

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

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

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.

symphotime_tips_and_tricks

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

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

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

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]]

symphotime64

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

2014/05/23 09:34		current
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-	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]].

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 ====

laser_intensity_response

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

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

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

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

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.

phasor_analysis

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

2019/08/28 18:15		current
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-	{{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~~

registering_new_scripts

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

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

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

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 =====

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) ======

privacy_policy

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

2019/12/03 16:45		current
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	Grundsätzlich besteht für die betroffene Person die Möglichkeit, einer Erfassung der durch den Matomo erzeugten, auf eine Nutzung dieser Internetseite bezogenen Daten zu widersprechen und eine solche zu verhindern. Hierzu muss die betroffene Person in Ihrem Browser "Do Not Track" einstellen.		Grundsätzlich besteht für die betroffene Person die Möglichkeit, einer Erfassung der durch den Matomo erzeugten, auf eine Nutzung dieser Internetseite bezogenen Daten zu widersprechen und eine solche zu verhindern. Hierzu muss die betroffene Person in Ihrem Browser "Do Not Track" einstellen.
-
-	Ferner besteht für die betroffene Person die Möglichkeit der anonymisierten Datenerhebung durch das Programm Matomo jederzeit mit Wirkung für die Zukunft zu widersprechen, indem sie auf den untenstehenden Link klicken. In diesem Fall wird ein sog. Opt-Out-Cookie im Browser der betroffenen Person abgelegt, was zur Folge hat, dass Matomo keinerlei Sitzungsdaten mehr erhebt. Wenn betroffene Person ihre Cookies löschen, so hat dies jedoch zur Folge, dass auch das Opt-Out-Cookie gelöscht wird und daher von den betroffene Person erneut aktiviert werden muss.

	Die Logs mit den Daten der betroffene Person werden nach spätestens 6 Monaten gelöscht.		Die Logs mit den Daten der betroffene Person werden nach spätestens 6 Monaten gelöscht.
-	{{url>https://www.picoquant.com/piwik/index.php?module=CoreAdminHome&action=optOut&language=en&backgroundColor=&fontColor=&fontSize=&fontFamily=}}

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

fluorescence_correlation_spectroscopy-_a_short_introduction

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

2023/02/17 13:43		current
Line 1:		Line 1:
	{{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 ======

asymptotic_standard_errors

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

2014/04/09 22:41		current
Line 5:		Line 5:
	The asymptotic standard errors (ASE) are used for analyzing fitting parameter error intervals. For illustration let's start at the best fit parameter set, which can be regarded as a single point in the parameter space. Now we remove the parameter for which we want to calculate the error intervals from this location, that is, we take a single step parallel to its parameter axis. We then calculate the reduced X<sup>2</sup> for this new paramter set. By iterating this procedure we get X<sup>2</sup> as a function of the parameter of interest. The intersection points of this function with a given X<sup>2</sup> confidence limit define the boundaries of the confidence interval of the parameter.		The asymptotic standard errors (ASE) are used for analyzing fitting parameter error intervals. For illustration let's start at the best fit parameter set, which can be regarded as a single point in the parameter space. Now we remove the parameter for which we want to calculate the error intervals from this location, that is, we take a single step parallel to its parameter axis. We then calculate the reduced X<sup>2</sup> for this new paramter set. By iterating this procedure we get X<sup>2</sup> as a function of the parameter of interest. The intersection points of this function with a given X<sup>2</sup> confidence limit define the boundaries of the confidence interval of the parameter.

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

reconvolution

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

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

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

residuals

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

2014/04/09 22:45		current
Line 13:		Line 13:
	$$w_i=\sqrt{D_i^{exp}}$$		$$w_i=\sqrt{D_i^{exp}}$$

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

tttr

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

2015/02/10 17:42		current
Line 6:		Line 6:
	The [[Products:TimeHarp 200]] (also the [[Products:TimeHarp 100]]), the [[Products:PicoHarp 300]] [[Products:HydraHarp 400]] support TTTR mode measurements. With the the [[Products:PicoHarp 300]] a slight shift in the nomenclature was introduced. Due to its capability of simultaneously recording both signal inputs (in interactive mode used as 'Start' and 'Stop') as two separate TTTR traces with a time resolution of 4ps, a new TTTR mode was introduced, the so called T2-mode. The traditional recording as with the [[Products:TimeHarp 200]] / [[Products:TimeHarp 100]] is called [[T3-mode]]. In [[T2-mode]] both signal inputs are functionally identical.		The [[Products:TimeHarp 200]] (also the [[Products:TimeHarp 100]]), the [[Products:PicoHarp 300]] [[Products:HydraHarp 400]] support TTTR mode measurements. With the the [[Products:PicoHarp 300]] a slight shift in the nomenclature was introduced. Due to its capability of simultaneously recording both signal inputs (in interactive mode used as 'Start' and 'Stop') as two separate TTTR traces with a time resolution of 4ps, a new TTTR mode was introduced, the so called T2-mode. The traditional recording as with the [[Products:TimeHarp 200]] / [[Products:TimeHarp 100]] is called [[T3-mode]]. In [[T2-mode]] both signal inputs are functionally identical.

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

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

calculate_ratiometric_single_pair_fret_distributions

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

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

	\\		\\

data_file_import

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

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

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

update

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

2023/02/23 07:24		current
Line 5:		Line 5:
	===== 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.

using_the_anisotropy_image_script

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

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

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

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

pycorrfit

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

2015/11/03 14:59		current
Line 4:		Line 4:
	"[...] 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]]

symphotime

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

2015/02/10 17:32		current
Line 3:		Line 3:
	The SymPhoTime software package is an integrated solution for data acquisition and analysis using PicoQuant's time-resolved confocal microscope MicroTime 200, LSM upgrade kits or TCSPC electronics.		The SymPhoTime software package is an integrated solution for data acquisition and analysis using PicoQuant's time-resolved confocal microscope MicroTime 200, LSM upgrade kits or TCSPC electronics.

-	After several very successful years [[software:SymPhoTime32]] has been replaced by the completely rewritten 64bit version [[software:SymPhoTim64]].	+	After several very successful years [[software:SymPhoTime32]] has been replaced by the completely rewritten 64bit version [[software:SymPhoTime64]].

configuring_symphotime64_after_installation

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

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

applications

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

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

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


Line 62:		Line 64:

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

lifetime_component_decomposition_phasor_plot_analysis

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

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

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

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

synchrotron_applications

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

2015/02/10 17:36		current
Line 9:		Line 9:
	* A cython library to access the original's manufacturer library from python and in a pythonic way,		* A cython library to access the original's manufacturer library from python and in a pythonic way,
	* An agent in the [[https://www.tango-controls.org/\|Tango distributed control system]] that manage the instrument,		* An agent in the [[https://www.tango-controls.org/\|Tango distributed control system]] that manage the instrument,
-	* A [[http://taurus-scada.org/\|Taurus gui]] with access to the instrument from the control system	+	* A [[http://taurus-scada.org/\|Taurus GUI]] with access to the instrument from the control system

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

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

novaflim_license

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

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

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

bdf

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

2015/02/10 17:44		current
Line 1:		Line 1:
	====== BDF ======		====== BDF ======

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

bifl

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

2014/04/09 22:38		current
Line 3:		Line 3:
	BIFL stands for Burst Integrated Fluorescence Lifetime: The photon events of a complete burst are accumulated into a [[TCSPC]] histogram which is used for lifetime analysis methods afterwards. Usually a burst is defined as a time interval for which the intensity of a time trace or [[MCS]] measurement is above a chosen threshold. Evaluation of all bursts present in an [[MCS]] trace, for example, results in a lifetime histogram.		BIFL stands for Burst Integrated Fluorescence Lifetime: The photon events of a complete burst are accumulated into a [[TCSPC]] histogram which is used for lifetime analysis methods afterwards. Usually a burst is defined as a time interval for which the intensity of a time trace or [[MCS]] measurement is above a chosen threshold. Evaluation of all bursts present in an [[MCS]] trace, for example, results in a lifetime histogram.

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

calculate_ratiometric_fret-images

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

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

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

calibrate_the_confocal_volume_for_fcs_using_the_fcs_calibration_script

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

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

determination_of_the_focal_width_with_the_focal

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

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

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

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

flim-fret_calculation_for_single_exponential_donors

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

2020/05/06 11:19		current
Line 85:		Line 85:

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

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

how_to_work_with_the_instrument_response_function_irf

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

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

intensity_time_trace_analysis

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

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

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

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

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_tcpsc_fitting_script

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

2023/09/08 00:37		current
Line 50:		Line 50:
	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_6.png?600 }}		{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_6.png?600 }}

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

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

lifetime-fitting_using_the_flim_script

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

2023/02/17 13:42		current
Line 92:		Line 92:

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

	For explanations about the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.\\		For explanations about the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.\\

lifetime-fitting_using_the_rapid_reconvolution_algorithm

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

2021/06/12 07:05		current
Line 1:		Line 1:
-		+	{{tag>howto tutorial FLIM analysis SymPhoTime MT LSM_upgrade reconvolution fitting}}
-	{{tag>howto tutorial FLIM analysis SPT SymPhoTime MT LSM_upgrade reconvolution fitting}}	+

roi_fitting_using_the_flim_script

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

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


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

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

separation_of_2_species_with

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

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

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

	====== Separation of 2 Species with Different Lifetimes Using FLCS ======		====== Separation of 2 Species with Different Lifetimes Using FLCS ======
Line 11:		Line 11:
	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 =====
Line 218:		Line 218:
	* Click on "Save result" to save this curve. Rename the generated file as "Cy5-FLCS".		* Click on "Save result" to save this curve. Rename the generated file as "Cy5-FLCS".
	* These curves can now be fitted by selecting "Transfer to fit". The process of how to fit a FCS curve is explained in the tutorial [[howto:Calculate and Fit FCS Traces with the FCS Script]].		* These curves can now be fitted by selecting "Transfer to fit". The process of how to fit a FCS curve is explained in the tutorial [[howto:Calculate and Fit FCS Traces with the FCS Script]].
-

visualizing_dynamics_with_the_multi_frame_flim_analysis

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

2015/11/03 15:33		current
Line 3:		Line 3:
	~~NOTOC~~		~~NOTOC~~

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

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

sepia_ii

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T15:04:30+00:00

2015/11/03 16:03		current
Line 3:		Line 3:
	The PDL 828 "Sepia II" is a high-end, multichannel driver for the picosecond pulsed laser diode heads of the LDH Series and LDH-FA Series as well as for the sub-nanosecond pulsed LEDs of the PLS Series. Wavelength changes are simply performed by plugging in a different laser or LED head.		The PDL 828 "Sepia II" is a high-end, multichannel driver for the picosecond pulsed laser diode heads of the LDH Series and LDH-FA Series as well as for the sub-nanosecond pulsed LEDs of the PLS Series. Wavelength changes are simply performed by plugging in a different laser or LED head.

-	The Sepia can either be controlled by the included software or by custom written software using the API. Demo Code utilising the API is available on [[https://github.com/PicoQuant/PQLaserDrv\|GitHub]].	+	The Sepia II can either be controlled by the included software or by custom written software using the API. Demo Code utilising the API is available on [[https://github.com/PicoQuant/PQLaserDrv\|GitHub]].

easytau

Anonymous (anonymous@undisclosed.example.com) — 2016-03-01T09:41:03+00:00

2016/03/01 10:40		current
Line 10:		Line 10:
	The easiest way to start with scripting measurements in EasyTau is to acquire a measurement with a wizard and then create a script from this measurement.		The easiest way to start with scripting measurements in EasyTau is to acquire a measurement with a wizard and then create a script from this measurement.

-	- Create a data set using a wizard of your choice	+	* Create a data set using a wizard of your choice
-	- Right click on the data set	+	* Right click on the data set
-	- Select ''Transfer script to customised mode, script page''	+	* Select ''Transfer script to customised mode, script page''
	{{ :software:create_script.png?600 \|}}		{{ :software:create_script.png?600 \|}}
-	- Adjust to your liking	+	* Adjust to your liking

supported_mt200_pc_configuration_for_symphotime_64

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

2023/02/17 13:44		current
Line 1:		Line 1:
-	{{tag>SymPhoTime MicroTime200 MicroTime100 Scanner PI Physik_Instrumente E725 E710 MadCityLabs Mad_City_Labs PC}}	+	{{tag>SymPhoTime MicroTime200 Scanner PI Physik_Instrumente E725 E710 MadCityLabs Mad_City_Labs PC}}
	~~TOC~~		~~TOC~~

symphotime_32

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

2026/04/23 19:16	current
Line 1:	Line 1:
	+	====== SymPhoTime 32 ======
	+	SymPhoTime 32 is the name of the now obsolete software package for data acquisition and analysis using PicoQuant's time-resolved confocal microscope [[products:MicroTime 200]], [[products:LSM upgrade kits]] or [[products:TCSPC electronics]].

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

dokuwiki

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

2026/04/23 19:16	current
Line 1:	Line 1:
	+	====== DokuWiki ======
	+
	+	[[doku>wiki:dokuwiki\|{{wiki:dokuwiki-128.png }}]] DokuWiki is a simple to use and highly versatile Open Source [[wp>wiki]] software that doesn't require a database. It is loved by users for its clean and readable [[wiki:syntax]]. The ease of maintenance, backup and integration makes it an administrator's favorite. Built in [[doku>acl\|access controls]] and [[doku>auth\|authentication connectors]] make DokuWiki especially useful in the enterprise context and the large number of [[doku>plugins]] contributed by its vibrant community allow for a broad range of use cases beyond a traditional wiki.
	+
	+	Read the [[doku>manual\|DokuWiki Manual]] to unleash the full power of DokuWiki.
	+
	+	===== Download =====
	+
	+	DokuWiki is available at https://download.dokuwiki.org/
	+
	+
	+	===== Read More =====
	+
	+	All documentation and additional information besides the [[syntax\|syntax description]] is maintained in the DokuWiki at [[doku>\|www.dokuwiki.org]].
	+
	+	About DokuWiki
	+
	+	* [[doku>features\|A feature list]] :!:
	+	* [[doku>users\|Happy Users]]
	+	* [[doku>press\|Who wrote about it]]
	+	* [[doku>blogroll\|What Bloggers think]]
	+	* [[https://www.wikimatrix.org/show/DokuWiki\|Compare it with other wiki software]]
	+
	+	Installing DokuWiki
	+
	+	* [[doku>requirements\|System Requirements]]
	+	* [[https://download.dokuwiki.org/\|Download DokuWiki]] :!:
	+	* [[doku>changes\|Change Log]]
	+	* [[doku>Install\|How to install or upgrade]] :!:
	+	* [[doku>config\|Configuration]]
	+
	+	Using DokuWiki
	+
	+	* [[doku>syntax\|Wiki Syntax]]
	+	* [[doku>manual\|The manual]] :!:
	+	* [[doku>FAQ\|Frequently Asked Questions (FAQ)]]
	+	* [[doku>glossary\|Glossary]]
	+
	+	Customizing DokuWiki
	+
	+	* [[doku>tips\|Tips and Tricks]]
	+	* [[doku>Template\|How to create and use templates]]
	+	* [[doku>plugins\|Installing plugins]]
	+	* [[doku>development\|Development Resources]]
	+
	+	DokuWiki Feedback and Community
	+
	+	* [[doku>newsletter\|Subscribe to the newsletter]] :!:
	+	* [[doku>mailinglist\|Join the mailing list]]
	+	* [[https://forum.dokuwiki.org\|Check out the user forum]]
	+	* [[doku>irc\|Talk to other users in the IRC channel]]
	+	* [[https://github.com/splitbrain/dokuwiki/issues\|Submit bugs and feature wishes]]
	+	* [[doku>thanks\|Some humble thanks]]
	+
	+
	+	===== Copyright =====
	+
	+	2004-2020 (c) Andreas Gohr <andi@splitbrain.org>((Please do not contact me for help and support -- use the [[doku>mailinglist]] or [[https://forum.dokuwiki.org\|forum]] instead)) and the DokuWiki Community
	+
	+	The DokuWiki engine is licensed under [[https://www.gnu.org/licenses/gpl.html\|GNU General Public License]] Version 2. If you use DokuWiki in your company, consider [[doku>donate\|donating]] a few bucks ;-).
	+
	+	Not sure what this means? See the [[doku>faq:license\|FAQ on the Licenses]].

phasor_plot_structure_separation

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

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

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

	Introduction		Introduction

create_time_gated_image

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

2020/10/14 14:10		current
Line 1:		Line 1:
-	//{{tag>howto analysis lifetime tutorial SymPhoTime}}//	+	{{tag>howto analysis lifetime tutorial SymPhoTime}}

	~~NOTOC~~		~~NOTOC~~

data_analysis

Anonymous (anonymous@undisclosed.example.com) — 2015-01-21T13:54:49+00:00

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

faq

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

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

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

measurement_hardware_instrumentation

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

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

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

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

novaflim_calculated_irf

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

2025/02/18 14:49		current
Line 1:		Line 1:
	====== NovaFLIM Calculated IRF ======		====== NovaFLIM Calculated IRF ======
		+	{{tag> faq software novaflim IRF}}
		+

	In the Nova Flim the IRF is parameterized as a combination of two Gaussian curves (a total of 5 fit parameters). These parameters are adjusted during Initial Fit.		In the Nova Flim the IRF is parameterized as a combination of two Gaussian curves (a total of 5 fit parameters). These parameters are adjusted during Initial Fit.

monte_carlo

Anonymous (anonymous@undisclosed.example.com) — 2014-04-09T20:45:42+00:00

2014/01/21 17:40		current
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-	PicoQuant Software makes use of Monte Carlo methods mainly as a means of finding initial values for [[fitting]] parameters before optimisation by a [[Marquardt-Levenberg]] algorithm. The principle is simple: A large number of random parameter sets is generated, for each one a [[chi square]] is calculated and the best one is taken as the initial parameter set.	+	PicoQuant Software makes use of Monte Carlo methods mainly as a means of finding initial values for [[wp>Regression_analysis\|fitting]] parameters before optimisation by a [[Marquardt-Levenberg]] algorithm. The principle is simple: A large number of random parameter sets is generated, for each one a [[chi square]] is calculated and the best one is taken as the initial parameter set.

	The second application is [[error analysis]], namely the [[bootstrap method]].		The second application is [[error analysis]], namely the [[bootstrap method]].

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.

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 =====

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 ====

measuring_quantum_yield

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

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

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

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

pattern_matching

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

2014/05/23 09:39		current
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	Pattern Matching Analysis by decomposing a recorded image into different user-defined patterns. Display of the calculated data using an RGB false color model. The calculated amplitudes of the first three defined patterns are depicted in three different colors for each pixel.		Pattern Matching Analysis by decomposing a recorded image into different user-defined patterns. Display of the calculated data using an RGB false color model. The calculated amplitudes of the first three defined patterns are depicted in three different colors for each pixel.
		+
		+	{{ youtube>L68IgRbNSLE?large }}

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

t3r_antibunching_-_slow_decay

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

2022/02/01 11:54		current
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	==== A decay caused by correlation statistics ====		==== A decay caused by correlation statistics ====
		+
		+	[{{howto:slope.png ?478x300\|Fig. 1: Baseline decay }}]
		+
	This decay is caused by correlation statistics. This happens because you correlate the first arriving photon against a photon at the longer time, which will be an increasingly unlikely event because the earliest stop photons always win and the experiment restarts.		This decay is caused by correlation statistics. This happens because you correlate the first arriving photon against a photon at the longer time, which will be an increasingly unlikely event because the earliest stop photons always win and the experiment restarts.

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

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

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.
-

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 ======

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

symphotime64

Anonymous (anonymous@undisclosed.example.com) — 2014-05-23T07:34:34+00:00

2026/04/23 19:16		current
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		+	This page is redirected to [[:software:symphotime64]].

fcs_viewer

Anonymous (anonymous@undisclosed.example.com) — 2015-02-25T16:51:37+00:00

2014/05/23 11:04		current
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-	[[https://github.com/dwaithe/FCS_viewer]]	+	[[https://github.com/paulmueller/FCS_viewer]]

	Contains a port of the PicoQuant Matlab demo code to Python.		Contains a port of the PicoQuant Matlab demo code to Python.

flimfit

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

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

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

supported_mt200_pc_configuration_for_symphotime_32

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

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

supported_mt200_pc_configuration_for_symphotime_64

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

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