TCSPC sequential recording

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. 

This difficulty is easily avoided by fluorescence lifetime detection. By using the sequential recording capability of multidimensional TCSPC, the fluorescence transients can be directly observed. A simple setup for recording the nonphotochemical quenching is shown in Fig. 5.31. 

Fig. 5.38 Stopped-flow measurement by TCSPC, triggered sequential recording, 100 ms per curve. Left Recorded sequence after a single stop. Right 500 stops accumulated. Data courtesy of Osman Bilsel, University of Massachusetts Medical School, Worcester, MA...

The sample (usually a cuvette) is measured from both sides under different polarisation angles. Two detectors and a router are used to detect Ip and L simultaneously [58, 59]. The T geometry with routed detection has twice the efficiency of a sequential measurement. Moreover, possible counting loss due to the dead time of the TCSPC module affects both channels in the same way and therefore does not affect the measured intensity ratio. The dual-detector routing technique is even able to record dynamic changes of the lifetime and depolarisation time. The drawback is that the instrument response functions of the two detectors are different,... 

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