Time-Resolved Fluorescence Wiki

pile-up_effect

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

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

asymptotic_standard_errors

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

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

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

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.

differential_count_rate

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

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

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

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

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

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]]
-

support_plane_analysis

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

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

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

chi_square_management

Anonymous (anonymous@undisclosed.example.com) — 2014-01-21T16:22:05+00:00

2013/08/09 09:30		current
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	====== Chi square ======		====== Chi square ======

-	The (normalised) χ<sup>2</sup> (read: chi square) is the optimisation parameter for [[least squares]] fitting (for a definition see there). For a perfect fit it should be near 1. As a measure of the goodness-of-fit it is insufficient. Other methods have to be used in addition, like examining the weighted [[residuals]] trace and the [[autocorrelation function]] of the weighted residuals trace.	+	The (normalized) χ<sup>2</sup> (read: chi square) is the optimization parameter for [[least squares]] fitting (for a definition see there). For a perfect fit it should be near 1. As a measure of the goodness-of-fit it is insufficient. Other methods have to be used in addition, like examining the weighted [[residuals]] trace and the [[autocorrelation function]] of the weighted residuals trace.

tttr

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

2015/02/10 17:42		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 [[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.

aotf

Anonymous (anonymous@undisclosed.example.com) — 2013-08-06T14:35:11+00:00

2013/08/06 16:34		current
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	$\sin\theta = \left (\frac{ m\lambda_l}{n\lambda_s} \right)$		$\sin\theta = \left (\frac{ m\lambda_l}{n\lambda_s} \right)$

-	where $m = ..., −2, −1, 0, 1, 2, ...$ is the order of diffraction. Diffraction from a sinusoidal modulation in a thin crystal solely results in the m = −1, 0, +1 diffraction orders.	+	where $m = ..., −2, −1, 0, 1, 2, ...$ is the order of diffraction. Diffraction from a sinusoidal modulation in a thin crystal solely results in the $m = −1, 0, +1$ diffraction orders.

	Additionally, the amount of light diffracted by the sound wave depends on the intensity of the sound signal. Hence, the intensity of the light in the diffracted beam can be modulated by sound intensity. Typically, the intensity of the m = 1 order can be varied between 0% and 80%.		Additionally, the amount of light diffracted by the sound wave depends on the intensity of the sound signal. Hence, the intensity of the light in the diffracted beam can be modulated by sound intensity. Typically, the intensity of the m = 1 order can be varied between 0% and 80%.

bifl

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

2014/04/09 22:38		current
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	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.

bootstrap

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

2014/01/21 17:41		current
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	Specifically, the bootstrap analysis takes a subset of N randomly chosen points out of the experimental data set, which itself consists of N data points. Since the points are chosen randomly, some of the points will be selected more than once; others will not be selected at all. Thus the 'simulated' data set is not identical to the original one, while the statistical properties of the original data set are maintained (since the number of data points remains the same).		Specifically, the bootstrap analysis takes a subset of N randomly chosen points out of the experimental data set, which itself consists of N data points. Since the points are chosen randomly, some of the points will be selected more than once; others will not be selected at all. Thus the 'simulated' data set is not identical to the original one, while the statistical properties of the original data set are maintained (since the number of data points remains the same).
-
-	At PQ the bootstrap method is exclusively used in [[FluoFit]]. For ensemble fitting as in [[FLIM]] it is -alas!- way too slow.
-

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

cfd

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

2013/08/06 16:37		current
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	[{{ glossary:constantfraction.png?400\|Comparison 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.

dead_time

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

2015/06/03 17:04		current
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	In TCSPC the term dead time refers to the time the TCSPC system needs armed again after detecting an event. During the dead time the TCSPC system is blind.		In TCSPC the term dead time refers to the time the TCSPC system needs armed again after detecting an event. During the dead time the TCSPC system is blind.

-	If for example two photons are detected with the dead time of the TCSPC device the second photon will be lost. This leads to the so called [[glossary:Pile Up Effect]].	+	If for example two photons are detected with the dead time of the TCSPC device the second photon will be lost. This leads to the so called [[glossary:Pile-up Effect]].

	Note that not only TCSPC devices but also photon counting devices exhibit dead times.		Note that not only TCSPC devices but also photon counting devices exhibit dead times.

hybrid_pmt

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

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

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

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