Time-Tag Recording

A variation of the TCSPC technique does not build up photon distributions but stores information about each individual photon. The mode is called time tag , time stamp , list , or FIFO mode. For each photon, the time in the signal period, the channel word, and the time from the start of the experiment, or macro time, is stored in a first-in-first-out (FIFO) buffer. During the measurement the FIFO is continuously read, and the photon data are stored in the main memory or on the hard disc of a computer. 

The structure in the time-tag mode is shown in Fig. 3.15. It contains the channel register, the time-measurement block, a macro time clock, and the FIFO buffer for a large number of photons. It has some similarity to the multidimensional TCSPC described in the paragraphs above. In fact, many advanced TCSPC modules have both the photon distribution and the time-tag mode implemented, and the configuration can be changed by a software command [25]. The sequencer then turns into the macrotime clock, and the memory turns into the FIFO buffer. 

When a photon is detected, the micro time in the signal period is measured by the time-measurement block. Simultaneously the deteetor ehannel number for the eurrent photon and often a number of additional bits from external experiment eontrol deviees are written into the channel register. The macro time clock delivers the time of the photon from the start of the experiment. All these data are written into the FIFO. 

The output of the FIFO is continuously read by the eomputer. Consequently, the time-tag mode delivers a continuous and virtually unlimited stream of photon data. It is, of eourse, imperative that the computer read the photon data at a rate higher than the average photon count rate. However, modem operation systems are multitask systems, and it is unlikely that the computer reads the FIFO eontinu-ously. Moreover, in typieal applications bursts of photons appear on a baekground 

The macro time clock can be started by an external experiment trigger or by a start-measurement command from the operating software. In some TCSPC modules the clock signal source of the macro time clock can be selected. The macro time clock can be an internal quartz oscillator, an external clock source, or the reference signal from the laser. Triggering and external clock synchronisation are absolute requirements for multimodule operation in the time-tag mode, see Sect. 5.11.3, page 189. 

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A different approach is event , time stamp", or time tag" recording. The technique writes the time of the individual detection events into memory. The general principle is shown in Fig. 2.11. A fast counter counts the clock periods from the moment when a pulse arrives at the trigger input. When a photon is detected the state of the counter is read and written into the next location of the memory. The memory is usually configured as FIFO (first in first out), i.e. the bytes at the output are read in the same order as they were written into the input. 

Time-tag recording means that the TCSPC channels do not build up a photon distribution but store each individual photon with its TAC time ( micro time ) and its time from the start of the experiment ( macro time ). The computer calculates the photon distribution at each location along the delay line and the time in the signal period. Time-tag recording of delay line data requires that the macro time clocks of all TCSPC channels be synchronised. Even then it is difficult to assign the data in the position channel to the correct data in the time channel. Due to slightly different CFD thresholds and different dead times, a photon recorded in the position channel need not necessarily be recorded in the time channel, and vice versa. To avoid misinterpretation of the data, a macro time resolution of 50 ns or finer is required. 

Spectroscopy of single molecules is based on fluorescence correlation, photoncounting histograms, or burst-integrated-lifetime techniques. Each case requires recording not only the times of the photons in the laser period, but also their absolute time. Modem time-resolved single molecule techniques therefore use almost exclusively the FIFO (time-tag) mode of TCSPC. The FIFO mode records all information about each individual photon, i.e. the time in the laser pulse sequence (micro time), the time from the start of the experiment (macro time), and the number of the detector that detected the photon (see Sect. 3.6, page 43). 

In typical TCSPC time-tag data, the clock period of the macro time, T, is shorter than the dead time of the TCSPC device. Therefore only one photon can be recorded at a particular macro time. Consequently, N t) and N(t + t) can only be 0 or 1. The multiplication in the autocorrelation function becomes a simple compare (or an exclusive-or) operation, and the integral of the autocorrelation becomes a shift, compare, and histogramming procedure. The calculation of FCS from TCSPC data is illustrated in Fig. 5.110. 

At first glanee eombining a pieoseeond eorrelation experiment with PCS looks simple. An antibunehing experiment eould be run in the PIPO mode, and the an-tibunehing and the PCS eurves be obtained from the miero times and macro times, respeetively. Unfortunately this approaeh has a flaw. Most of the photons emitted by the sample eause either a start without a stop, or a stop without a start however, the TCSPC module reeords only eomplete start-stop events. Therefore the system records only a tiny fraction of the photons reaehing the detectors. The low effieiency makes the obtained time-tag data praetieally useless for PCS. 

A mueh higher burst resolution ean be obtained by reeording the photons in the FIFO or time-tag mode. The time-tag mode is deseribed under Sect. 3.6, page 43. From the time-tag data, BIFL results with a burst resolution down to the laser pulse period ean be obtained. MCS traees are available, and FCS and PCHs can be ealeulated. Beeause the full information about all photons is recorded time-tag data are extremely flexible. Conformational dynamics, rotational relaxation, and intersystem erossing ean be investigated at almost any time scale [108, 154, 155, 295, 419, 500]. However, time-tag data are also voluminous. For each photon four or six bytes are reeorded, and file sizes of a gigabyte per measurement are not unusual. 

These molecules are then brought into the focus and time-tag data are acquired. From the macro times of the recorded photons traces of the emission intensity of a single molecule are built up. The traces show bright periods, when the molecule cycles between the ground state and the excited singlet state, and dark periods, when the molecule is in the triplet state (Fig. 5.130). 

The afterpulsing probability can be measured by illuminating the detector by a source of continuous classic light, such as an incandescent lamp or an LED, and recording the detector pulses in the FIFO (time-tag) mode of a TCSPC module. 

Fig. 1.18 A film of silicone oil of 1 mm thickness is flowing along a vertically oriented planer sheet of PMMA. In a tagging experiment, a horizontal slice of 2 mm thickness is marked and its deformation is recorded as a function of the separation time A between the...

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

For the analysis in which the correlation function has to be recorded in the ns regime a typical Flanbury Brown and Twiss [18] set up. Figure 4.4 shows a confocal setup (discussed later) in combination with a beam splitter and a time to amplitude converter (TAG). Since both pathways for the emitted photons acting as start or stop for the time amplitude converter (TAG) are equivalent the correlation curves registered in a multichannel analyzer (MGA) are symmetric relative to 0 time. 

Typical records obtained from slurries are shown in Figs. 6.26 and 6.27. The variation of the signal profile can be explained by different flow models (Porges, 1984). The averaged flow velocity can be derived from the PNA signal recorded by the detector at a known distance downstream from the PNA source. The PNA velocity results agreed with timed-diversion measurements to within 0.5% and no systematic deviation was found. We believe that the accuracy achieved by the PNA technique can be better than that obtained by other techniques because the PNA system irradiates the entire duct, and the shape of the readout is directly related to the motion of the tags. 

Fig. 7.75 Recording of the same signal as in Fig. 7.74, but with a TAG gain of 10. The time interval of the signal is stretched over the full ADC range, resulting in an increase of the available number of time channels and a recorded width of the peak of 72.4 ns...

The situation for reversed start-stop and high repetition rate signals is shown in Fig. 7.85. The TAG is started when a photon is detected and stopped with the next laser pulse. Within the time between the start and the stop, the TAG is unable to record a second photon. The resulting loss is the classic pile-up effect. 

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