Time-Resolved Fluorescence Wiki

t3-mode

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

2015/02/10 16:26		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.

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	If the counter 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. The 12 bits for the start-stop time difference cover a time span of 4096*R where R is the chosen resolution. At the highest possible resolution of 4 ps this results in a span of 16 ns. If the time difference between photon and the last sync event is larger, the photon event cannot be recorded. This is the same as in histogramming mode, where the number of bins is also finite. However, by choosing a suitable sync rate and a compatible resolution R, it should be possible to reasonably accommodate all relevant experiment scenarios. R can be chosen in doubling steps between 4 ps and 512 ps.		If the counter 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. The 12 bits for the start-stop time difference cover a time span of 4096*R where R is the chosen resolution. At the highest possible resolution of 4 ps this results in a span of 16 ns. If the time difference between photon and the last sync event is larger, the photon event cannot be recorded. This is the same as in histogramming mode, where the number of bins is also finite. However, by choosing a suitable sync rate and a compatible resolution R, it should be possible to reasonably accommodate all relevant experiment scenarios. R can be chosen in doubling steps between 4 ps and 512 ps.

-	Dead time in T3 Mode is the same as in the other modes (eg. typ. 95 ns for PicoHarp) Since there is only one photon channel, only autocorrelations can be calculated (unless a router is used). This can be done at the resolution R but of course only starting from lag times larger than the deadtime.	+	[[Dead time]] in T3 Mode is the same as in the other modes (eg. typ. 95 ns for PicoHarp) Since there is only one photon channel, only autocorrelations can be calculated (unless a router is used). This can be done at the resolution R 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 th 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 is fully available for input channel 1. For all practically relevant fluorescence detection applications this 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 with Windows 2000 or XP a sustained average count rate over 4 Mcps is possible. This total transfer rate is fully available for input channel 1. For all practically relevant fluorescence detection applications this is more than sufficient.

	For maximum throughput, T3 Mode data streams are normally written directly to disk. The current Harp software does not provide any immediate data visualization during a T3 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 Harp software installation CD contains demo programs showing how T3 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.		For maximum throughput, T3 Mode data streams are normally written directly to disk. The current Harp software does not provide any immediate data visualization during a T3 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 Harp software installation CD contains demo programs showing how T3 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.

	see also [[t2-mode]]		see also [[t2-mode]]

cfd

Anonymous (anonymous@undisclosed.example.com) — 2015-09-23T14:55:11+00:00

2013/08/06 14:37		current
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	CFD stands for Constant Fraction Discriminator.		CFD stands for Constant Fraction Discriminator.

-	[{{ glossary:constantfraction.png?400\|Comparation of threshold activation (left) to CFD activation (right) }}]	+	[{{ glossary:constantfraction.png?400\|Comparison of threshold activation (left) to CFD activation (right) }}]

-	The CFD is used to extract precise timing information from electrical detector pulses that may vary in amplitude. Thereby the overall system IRF can be narrowed and some of the random background signal can be suppressed. This cannot not be achieved with a simple threshold detector (comparator). Constant fraction discrimination is very important, especially in the case of [[PMTs]], because their pulse amplitudes vary significantly.	+	The CFD is used to extract precise timing information from electrical detector pulses that may vary in amplitude. Thereby the overall system IRF can be narrowed and some of the random background signal can be suppressed. This cannot not be achieved with a simple threshold detector (comparator). Constant fraction discrimination is very important, especially in the case of [[PMT]]s, because their pulse amplitudes vary significantly.

	A CFD compares the original detector signal with an amplified and delayed version of itself. The signal derived from this comparison changes polarity exactly when a constant fraction of the detector pulse height is reached. The zero crossing point of this signal is therefore suitable to derive a timing signal independent from the amplitude of the input pulse. This is done by comparing this signal to a zero cross level. This level should be adjusted to remove events that originate from signal noise. CFDs furthermore permit to set a discriminator threshold for the pulse amplitude.		A CFD compares the original detector signal with an amplified and delayed version of itself. The signal derived from this comparison changes polarity exactly when a constant fraction of the detector pulse height is reached. The zero crossing point of this signal is therefore suitable to derive a timing signal independent from the amplitude of the input pulse. This is done by comparing this signal to a zero cross level. This level should be adjusted to remove events that originate from signal noise. CFDs furthermore permit to set a discriminator threshold for the pulse amplitude.

deconvolution

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

2013/08/06 12:34		current
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	====== Deconvolution ======		====== Deconvolution ======
-
-	===== What is "Deconvolution"? =====

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

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


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

flim

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

2015/11/03 14:28		current
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		+
	{{tag>FLIM}}		{{tag>FLIM}}
		+
		+	~~TOC~~
	====== FLIM ======		====== FLIM ======

t2-mode

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

2013/08/06 14:59		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]]



-	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 times 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 deadtime.	+	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 th 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]]
-

tttr

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

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

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

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