TCSPC advanced

The introduction and diversification of genetically encoded fluorescent proteins (FPs) [1] and the expansion of available biological fluorophores have propelled biomedical fluorescent imaging forward into new era of development [2], Particular excitement surrounds the advances in microscopy, for example, inexpensive time-correlated single photon counting (TCSPC) cards for desktop computers that do away with the need for expensive and complex racks of equipment and compact infrared femtosecond pulse length semiconductor lasers, like the Mai Tai, mode locked titanium sapphire laser from Spectra physics, or the similar Chameleon manufactured by Coherent, Inc., that enable multiphoton excitation. 

For example, optical detection techniques are inereasingly used for experiments on biological samples. The limited photostability of these samples sets severe limitations to the excitation power. The photon rates obtained from most biological samples are well within the counting capability of advanced TCSPC. Under these conditions TCSPC yields acquisition times shorter than any analog technique. 

Compared with the very limited capacity of the more or less discrete control electronics of classic TCSPC systems, the use of FPGAs led to a breakthrough in functionality. Advanced TCSPC devices use a multidimensional histogramming process. They record the photon density not only as a function of the time in the signal period, but also of other parameters, such as wavelength, spatial coordinates, location within a scanning area, the time from the start of the experiment, or other externally measured variables. It is actually the multidimensionality and flexibility in the data acquisition process that makes new TCSPC techniques really advanced . 

A variation of the TCSPC technique does not build up photon distributions but stores information about each individual photon. This is called time tag , time stamp , list , or FIFO mode. The memory is configured as a FIFO buffer. For each photon, this method stores the time in the signal period ( micro time ), the time from the start of the experiment ( macro time ), and the data word at the channel input. During the measurement, the FIFO is continuously read, and the photon data are transferred into the main memory or to the hard disc of a computer. Advanced TCSPC devices often can be configured either to build up a multidimensional photon distribution or to store the individual photons. The FIFO mode is described in Sect. 3.6, page 43. 

Of course, the multidetector technique does not increase the maximum throughput rate of a TCSPC system. In any TCSPC device there is a small but noticeable loss of photons due to the dead time" of the processing electronics. The dead time of advanced TCSPC devices is of the order of 100 ns, and for count rates above 1 MHz the counting loss becomes noticeable (see Sect 7.9, page 332). The counting loss for a multidetector TCSPC system is the same as for a single detector system operated at the total count rate of the detectors of a multidetector system. 

Because the sequence is controlled by the TCSPC hardware, it is possible to achieve extremely fast and accurate stepping, down to less than a microsecond per data block. Of course, sequential recording can also be achieved by software control of a TCSPC device, and advanced TCSPC devices in fact include operating modes for recording software-controlled sequences. However, modem computers are far from being real-time systems. Stepping faster than 100 ms per step becomes inaccurate, which makes an accumulation of software-controlled sequences impossible. 

The timing pulse from the multichannel plate is processed in the usual way in the time-measurement block. The pulses from the anode structure of the detector, A1 through A4, are converted by four ADCs on the TCSPC board. The position of the photon is calculated in a digital arithmetic unit. The unit has to deliver one X Y data pair within 100 ns or less to keep the dead time within the common standard of advanced TCSPC. The solution to the problem is pipelining ... 

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. 

Compared with classic systems, the dead time of advanced TCSPC systems has been considerably reduced. It is however, still on the order of 100 to 150 ns. The fraction of photons lost in the dead time - the counting loss - becomes noticeable at detector count rates higher than 10% of the reciprocal dead time (see Sect. 7.9.2, page 338). The counting loss can be compensated for by a dead-time-compensated acquisition time. Therefore, often a relatively high loss can be tolerated. The practical limit is the maximum useful" count rate, which is defined as the recorded rate at which 50% of the photons are lost. For currently available TCSPC modules, the maximum useful count rate ranges from 3 to 5 MHz, corresponding to a detector count rate from 6 to 10 MHz. 

Advanced TCSPC devices usually have spectrum-scan modes that reeord several spectra in different time windows simultaneously. The prineiple is shown in Fig. 5.20. The wavelength is scanned, and for each wavelength a fluorescence decay curve is recorded. The counts in the time channels of the deeay eurve are averaged within selectable time intervals. The averaged counts are stored as functions of the wavelength. Several independent time windows can be used simultaneously. Therefore the efficiency is better than for a system that uses a single window discriminator. 

Advanced TCSPC techniques can reduce the size and the weight of the instra-ment considerably. As shown in Fig. 5.49, the detector signals are divided into four groups of eight signals and connected to four routers. The routers are connected to individual channels of a four-channel TCSPC system. The TCSPC system is then reduced to the size of an industrial or even a standard PC. 

Recently Liebert et al. have demonstrated that advanced TCSPC is able to record effects of brain activity with 50 ms time resolution, clear separation of scattering and absorption, and probably better depth resolution than CW or frequency-domain techniques [324, 327, 328]. A system of four parallel TCSPC modules with four individual detectors and several multiplexed laser diode lasers is used. A fast sequence of time-of-flight distributions is recorded in consecutive time intervals of 50 to 100 ms. Variations of the optical properties in the brain are derived from the intensity and the first and second moments of the time-of-flight distributions [325]. 

This is exactly what advanced multidimensional TCSPC is capable of. The scanning technique of advaneed TCSPC (see page 37) therefore almost perfectly fits the laser seanning mieroseope. 

Advanced TCSPC techniques provide several easy and reliable ways of lifetime imaging with scan stages (see Sect. 3.4, page 37). 

In combination with advanced TCSPC, the systems can be used to record fluorescence decay curves, dynamic changes of fluorescence decay curves, fluorescence correlation in combination with fluorescence lifetime, and spectrally resolved fluorescence decay profiles. Examples for dynamic lifetime measurements and spectrally resolved lifetime measurements by a multianode PMT with routing are shown under Sects. 5.4.1, page 90, and 5.2, page 84. 

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. 

A TCSPC oscilloscope mode is implemented in most advanced TCSPC modules. The mode has become an indispensable tool for a large number of technical jobs. Alignment and optimisation of optical systems often requires not only maximising the efficiency but also localising and removing optical reflections, leakage of excitation light, or pulse dispersion. 

To obtain position sensitivity, the single anode can be replaced with an array of individual anode elements [297, 298] see Fig. 6.4. The position of the corresponding photon on the photocathode can be determined by individually detecting the pulses from the anode elements. Multianode PMTs are particularly interesting in conjunction with the multidetector capability of advanced TCSPC techniques. 

In advanced TCSPC systems external wiring is reduced to a minimum. Nevertheless finding the right cables, connectors and adapters can be a nightmare. The most common connections systems currently used are BNC, SMA, SMB, MCX, and LEMO connectors. The connectors are shown in Fig. 7.46. 

Due to the finite speed of signal processing, a photon eounter is unable to deteet a seeond photon within a certain dead time after the deteetion of a previous one. For gated photon counters or multichannel scalers, the dead time ean be as short as a nanoseeond. The relatively complicated signal proeessing sequenee in a TCSPC deviee leads to a much longer dead time. Older TCSPC deviees had dead times of the order of 10 ps. Newer, more advanced TCSPC modules are mueh faster but still have a dead time in the range of 100 to 150 ns. 

Advanced TCSPC techniques have resulted in a number of spectacular applications in different fields of time-resolved spectroscopy. Nevertheless, a large number of potential applications clearly could benefit from TCSPC but do not use or do not fully exploit the capabilities of the currently available techniques and devices. This may be due in part to the continuing misperception that TCSPC is unable to reeord high photon rates, to achieve short acquisition times, or to reveal dynamie effeets in the fluorescence or scattering behaviour of the systems investigated. Another obstacle may be that TCSPC users often do not take the effort to understand the advanced features of the technique and consequently do not make the most effieient use of the devices they have. 

TCSPC techniques also have enormous potential in drug screening and in DNA analysis by spectroscopic techniques. It is commonly believed that the data throughput of TCSPC is insufficient for these applications. However, as with many other applications, it is likely that the bottleneck is the photostability of the sample rather than the throughput of TCSPC. Thus advanced TCSPC techniques, especially combinations of multidetector and multimodule techniques, appear likely to be used in this field. 

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