Principles of TCSPC

A eloser look at the technical principles of TCSPC and their applieation to experiments beyond the traditional fluorescence lifetime measurements reveals quite a different picture now. 

Fig. 3.2 Principle of TCSPC multidetector operation. The detectors are receiving different signals originating from the same excitation laser. The photon pulses from both detectors are combined, and the times of the pulses are measured in a single TAC. A routing signal indicates which of the detectors detected the currently processed photon. The TCSPC module puts the photons from different detectors into different memory segments...

However, almost all advanced TCPCS devices are able to reeord lifetime data and PCS data simultaneously [25, 65]. The advantage compared to the traditional approach is that PCS and lifetime data originate from the same sample, from the same spot of a sample, or even from the same molecules. TCSPC data can therefore be used to distinguish between different types of molecules, different quenching states, or different binding or conformation states of dye-protein eomplexes it is also possible to include lifetime variations in the correlation [498, 548]. The principle of TCSPC-based PCS is shown in Pig. 5.109. 

The principles of TCSPC can be understood by examina- Prior to examining the electronic components in more don of lui instrument schematic (Figure 4.7). The experi- detail, it is valuable to examine the actual data. Anintensity... 

Figure 11.13 Representation of fluorescence decay and of the principle of the measurement. On the graph, each point represents the contents of a memory channel (coupling time/numher of photons). The exponential curve of decay appears here in the form of a straight line. This is due to the choice of a logarithmic scale for the ordinate. On the right, a representation of the TCSPC (time-correlated single-photon counting).

The routing capability of TCSPC can be used to multiplex several light signals and record them quasisimultaneously. The principle of multiplexed TCSPC is... 

There are a number of different time measurement teehniques applicable to TCSPC. The TAC-ADC principle of the elassic TCSPC teehnique has been upgraded with a fast, error-cancelling ADC technique. Integrated eireuits for direct time-to digital conversion (TDCs) have been developed, and a sine-wave time-eonversion teehnique has been introdueed. 

In TCSPC the principle of dithering is reversed. The ADC input signal is random, and a determinate dither signal is used. The principle is shown in Fig. 4.7. 

The intensities of classic lifetime experiments are well within the TCSPC range. Furthermore, sensitivity, time resolution, and accuracy are often more important than short acquisition time. Therefore many classic lifetime systems still use the classic NIM-based TCSPC technique. The general principles of fluorescence lifetime experiments are described in [308, 389], and various fluorescence lifetime spectrometers are commercially available. 

The stopped-flow technique uses the same mixing cell as described for the continuous flow (Fig. 5.35). However, the flow of the reagents is periodically stopped, and the reaction is observed by recording the transient changes in absorption or fluorescence intensity. The principle of a TCSPC-based detection system for stopped flow is shown in Fig. 5.37. An overview about the technique is given in [440]. 

Fig. 5.37 Principle of a stopped-flow instrument with TCSPC...

The principle of a typical TCSPC-based scanning mammograph is shown in Fig. 5.44. 

The common use of fluorescence in small-animal imaging makes time-resolution almost mandatory. As for the other DOT applications, time-resolved small-animal imaging is performed by modulation techniques and by TCSPC. TCSPC has the benefit that it is able to resolve complex decay functions. The principle of a typical time-resolved instrument [174] is shown in Fig. 5.57. 

The term microfluorometer or microspectrofluorometer is used for systems that excite a small, usually diffraction-limited volume of a sample under a microscope and record the fluorescence, either with wavelength resolution or without. The borderline with FLIM techniques is not clearly defined. A TCSPC FLIM system can be used to record the fluorescence of a single point, and a microspec-trofluorometer combined with a scanning stage can be used as a FLIM system. Some typical principles of microfluorometry are shown in Fig. 5.97. 

A considerable improvement can be expected from the application of TCSPC to autofluorescence imaging of tissue. As mentioned in the application section of this book, the fluorescence lifetime helps not only to separate the different types of fluorescence but also to characterise the state of their binding to proteins, lipids, or DNA, as well as the oxygen saturation or the pH of the tissue. The first steps have been taken in time-resolved two-photon microscopy. The same basie optieal principles are leading to imaging of macroscopic samples. 

Technically, the photons of all detectors are combined into a common timing pulse line. Simultaneously, a detector number signal is generated that indicates in which of the detectors a particular photon was detected. The photon pulses are sent through the normal time measurement procedure of the TCSPC device. The detector numbers are used as a channel (or routing) signal for multidimensional TCSPC, routing the photons from the individual detectors into different waveform memory sections. The principle is illustrated in Fig. 3.2. 

In principle, parallel imaging ean be eombined with multiplexing and sequencing. Of course, the size (or pixel number) of the images, sequeneer steps, and multiplexing channels that can be used simultaneously are limited by the memory space in the TCSPC module. 

Routing the photons by the ADC result of a second TCSPC channel faces similar problems. It must be guaranteed that the position information is derived from the same photon as the time information. This is possible by gating the start CFD of one TCSPC channel with the detection of a photon in the other. The principle is shown in Fig. 3.14. 

Because of its superior time resolution the conventional TAC-ADC technique used in the classie TCSPC setups is still used in advaneed TCSPC devices. However, the conventional TAC-ADC teehnique has been upgraded by a modified ADC principle that cancels the nonuniformity of the ADC eharacteristics. With the new ADC technique, ADC ehips with moderate aeeuraey but extremely high speed can be used. Together with a speed-optimised TAC eireuitry, the new ADC technique achieves exceptionally high conversion rates. 

Currently TCSPC modules based on the TAC-ADC principle use 12-bit ADCs, i.e. resolve the recorded waveform into 4096 time channels. Many applications do not require this large a number of time channels. It is more important to have a large number of waveform memories available, and therefore desirable to reduce the number of time channels. Electronically it is relatively simple to bin several of the original ADC channels into one time channel of the recorded photon distribution, see Fig. 4.9. 

A third time-conversion technique uses sine-wave signals for time measurement. Two orthogonal sine-wave signals are sampled with the start and the stop pulses. The phase difference between start and stop is used as time information [313]. Currently the sine-wave technique is inferior to the TAC-ADC principle and the TDC principle in terms of count rate. It is not used in single-board TCSPC devices. However, with the fast progress in ADC and signal processor speed the sine wave technique may become competitive with the other techniques. The principle is shown in Fig. 4.16. 

The lasers must then be multiplexed at a rate faster than the changes expected in the sample. One way to multiplex lasers is to synchronise their pulse periods and delay the pulses of different lasers by different fractions of the pulse period. The fluorescence signals are recorded simultaneously in the same TAC range of a TCSPC device. The principle is shown in Fig. 5.26. 

In principle, the recorded sequence could be triggered with the stimulation event and accumulated. However, in practice there are is a strong variation in the data due to heart beat [197, 502] and respiration. The response of the brain to the stimulation can more reliably be separated from other effects by recording the full sequence over a large number of stimulation events. To record a virtually unlimited sequence the TCSPC channels are operated in the continuous flow mode (see Fig. 3.9, page 36). 

The setup shown in Fig. 5.49 can, in principle, be used to record fast changes in the brain at 4 laser wavelengths and 32 detector positions. However, the limited speed of the fibre switch normally allows one to record sequences only for one or two source positions at a time. The result is a total number of 128 to 256 waveforms each 50 to 100 ms or 32 to 64 per TCSPC module. The corresponding readout rate in the memory swapping mode is well within the range of currently used TCSPC modules. However, improved fibre switches may allow one to multiplex a larger number of source positions at a rate of 100 s" or faster. The data transfer rate then exceeds 10 Mbyte/s, and precautions have to be taken to sustain this rate over a longer time. 

The principle is shown in Fig. 5.100. The investigated light signal is split by a 1 1 beam splitter, and the two light signals are fed into separate detectors. One detector delivers the start pulses, the other the stop pulses of a TCSPC device. The stop pulses are delayed by a few ns to place the coincidence point in the centre of the recorded time interval. The setup delivers a histogram of the time differences between the photons at both detectors. Because separate detectors are used for start and stop, there is no problem with detector dead time. 

The principle shown in Fig. 5.100 ean be extended to more than two start detectors. The photons can be separated depending on their wavelength or polarisation. The photons of different channels ean be used as start ehannels and be correlated with the photons in a common stop channel. The TCSPC system uses a router to combine the events in the start ehannels into a eommon timing pulse line, see Fig. 5.104. 

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