TCSPC time measurement

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).

Time-resolved PL measurements were carried out using time-correlated singlephoton counting (TCSPC). Time-integrated PL and EL measurements were performed using the TCSPC apparatus in time-integrated mode. The temperature was controlled using an Oxford Instruments OptistatCF Helium cryostat. 

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. 

Figure 3.4 shows how the router works in concert with the TCSPC module. The CFD of the TCSPC module receives the single-photon pulse from the router, i.e. the amplified pulse of the detector that detected the photon. When the CFD detects this pulse, it starts a normal time measurement sequence for the detected photon. Furthermore, the output pulse of the CFD loads the channel information from the router into the channel register. The latched channel information is used as a dimension in the multidimensional recording process. In other words, it controls the memory block in which the photon is stored. Thus, in the TCSPC memory separate photon distributions for the individual detectors build up. In the simplest case, these photon distributions are single waveforms. However, if the sequencer is used, the photon distributions of the individual detectors can be multidimensional themselves. 

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. 

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. 

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. 

Acquisition times for TCSPC FLIM measurements can vary widely. In vivo lifetime measurements of the human ocular fundus in conjunction with an ophthalmic scanner delivered single exponential lifetimes for an array of 128 x 128 pixels within a few seconds [451, 452, 454]. High-quality double exponential lifetime images of microscopic samples were obtained within 10 seconds by a four-module TCSPC system (see Fig. 5.84) [39]. On the other hand, for the double exponential decay data of FRET measurements in live cells (see Fig. 5.87), acquisition times ranged from 5 to 30 minutes [32, 37]. In practice the acquisition time depends on the size and the photostability of the sample and the requirements for accuracy rather than on the counting capability of the TCSPC device. 

An example of a measurement obtained by a delay-line MCP and two TCSPC cards is shown in Fig. 5.98. One TCSPC card measures the delay of the photon pulses between the outputs of the delay line, i.e. the position of the photon in the fluorescence spectrum. The second card measures the times of the photons in the decay curve. It receives a position-proportional routing signal from the first card and thus builds up the photon distribution over time and wavelength, see Fig. 3.14, page 42. 

The TCSPC-FCS technique can also be used in conjunction with a continuous laser. Of course, in this case the measurement does not deliver a meaningful miero time, and no lifetime data are obtained. Because the TCSPC module needs a synchronisation pulse to finish the time measurement for a recorded photon, an artificial stop pulse must be provided. This can be the delayed detector pulse itself or a signal from a pulse generator see Fig. 5.116. 

Figure 5.144 shows an example of a TCSPC oseilloseope measurement. The fluoreseenee of chlorophyll in a leaf was reeorded at a eount rate of 410 photons per second and an acquisition time of 100 ms. The time ehannel width was 9.8 ps. The fluoreseenee was exeited by a diode laser at a wavelength of 650 nm and a repetition rate of 50 MHz. 

The time measurement block of a TCSPC device working in the reversed start-stop mode is shown in Fig. 7.69. 

Fig. 7.69 TAC control parameters in the time measurement block of TCSPC.

Fig. 3.1. Schematic diagram of a TCSPC setup. Using a fast timing device (e.g., time-to-amplitude-converter) the time is measured between the excitation pulse and the detection of a photon. By repeating this procedure many times a decay curve is measured. TAC time to amplitude convertor, MCA multi channel analyzer, PC personal computer. 

In contrast, time domain instruments attempt to directly measure the decay characteristics of a fluorophore of interest by excitation with ultrashort light pulses and monitoring the decay using either TCSPC [5] or a time gated image intensifier [8],... 

Our objective was to probe fluorescence over a time domain as large as possible. To this end we combined two different detection techniques, FU and TCSPC, allowing us to perform measurements from 100 fs to hundreds of nanoseconds. Notably, we use the same laser excitation source the third harmonic of a titane sapphire laser (267 nm, 100 fs). This is important because the excited state population is created under identical conditions in the two types of experiments. The time-resolution obtained after deconvolution of the recorded signals is 100 fs and 10 ps for FU and TCSPC, respectively. For reasons explained below, FU only detects emission corresponding to highly allowed transitions. TCSPC, on the other hand, is capable to monitor not only allowed but also very weak or forbidden transition. Therefore, particular care must be taken when merging data obtained by these two techniques as described in Ref. 10. 

To further characterize the mobility of the IRE loop, time-resolved isotropic fluorescence emission decay components of the IRE RNAs were determined as a function of temperature. Some details of the measurements and data assessment will be necessary here to appreciate both the utility of the information and caveats about its literal interpretation. Considering first the TCSPC instrument itself, some uncertainty in the measurements arise from its intrinsic parameters. With 300 nm incident light, the IRF of the photomultiplier tube ranged from 190 to 276 ps full-width at half-height (FWHH). The width of the IRF and the time resolution (32.5 ps/channel) limit the short components that can be reliably extracted from the fit, and certainly those <200 ps will have large errors on their amplitudes and lifetimes. Fluorescence emission decay components as short as 9—20 ps (Larsen et al., 2001) and 30—70 ps (Guest el al., 1991) (and much shorter by Wan et al., 2000) have been measured for 2AP in a stacked conformation, but in our instrument, a fit to such a short lifetime would be inaccurate. 

Three techniques are actually available for measuring the fluorescence lifetime Strobe, Time Correlated Single Photon Counting (TCSPC), and multifrequency and crosscorrelation spectroscopy. Strobe and TCSPC are based on measurement in the time domain, while multifrequency and cross-correlation spectroscopy measure fluorescence lifetimes in the frequency domain. The time domain allows direct observation of fluorescence decay, while the frequency domain is a more indirect approach in which the information regarding the fluorescence decay is implicit. 

In the time-correlated single-photon counting (TCSPC) technique, the sample is excited with a pulsed light source. The light source, optics, and detector are adjusted so that, for a given sample, no more than one photon is detected. When the source is pulsed, a timer is started. When a photon reaches the detector, the time is measured. Over the course of the... 

Figure 15-11. (a) Steady-state absorption and fluorescence spectra of TMC and cytosine in aqueous solution pH 7 at 25°C. The steady-state fluorescence spectra of the two compounds were measured using solutions having the same absorbance (ca. 0.3) at the wavelength of excitation (280 nm). (b) Observed (dot) and fitted (solid) fluorescence decay of TMC at 350 nm after excitation at 280 nm. Also shown is die temporal response function and the time-resolution of the TCSPC system estimated to be 30ps after deconvolution. (Reprinted with permission from Ref. [23].)... 

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