TCSPC counting

In Fig. 3.5A a comparison between time-gated detection and TCSPC is shown. The time-gated detection system was based on four 2 ns wide gates. The first gate opened about 0.5 ns after the peak of the excitation pulse from a pulsed diode laser. The TCSPC trace was recorded using 1024 channels of 34.5 ps width. The specimen consisted of a piece of fluorescent plastic with a lifetime of about 3.8 ns. In order to compare the results, approximately 1700-1800 counts were recorded in both experiments. The lifetimes obtained with TG and TCSPC amounted to 3.85 0.2 ns and 3.80 0.2 ns respectively, see Fig. 3.5B. Both techniques yield comparable lifetime estimations and statistical errors. 

In time-gated photon counting, comparatively high photon count rates can be employed count rates as high as 10 MHz are often used. TG has the advantage of virtually no dead-time of the detection electronics ( 1 ns), whereas the dead-time of the TCSPC electronics is usually on the order of 125-350 ns. This causes loss of detected photons, and a reduced actual photon economy of TCSPC at high count rates. 

Not only PMTs and other detectors such as avalanche photodiodes suffer from dead-time effects also the detection electronics may have significant dead-times. Typical dead-times of TCSPC electronics are in the range 125-350 ns. This may seriously impair the efficiency of detection at high count rates. The dead-time effects of the electronics in time-gated single photon detection are usually negligible. 

In wide field microscopy, spatial information of the entire image is acquired simultaneously thus providing comparatively short acquisition times compared with scanning microscopy implementations. Combining TCSPC with wide field microscopy is not straightforward. However, a four quadrant anode multichannel plate (MCP) has been used for time- and space-correlated SPC experiments [25, 26]. This detector has excellent timing properties that make it very suitable for FLIM. Unfortunately, it can be operated only at low count-rates ( 105-106 Hz) therefore, it requires comparatively long acquisition times (minutes). 

Because of the low timing-jitter (down to 25 ps) TCSPC-based systems are often equipped with a MCP-PMT at detriment of acquisition speed (<106 counts per second). On the other hand, a TG-SPC system equipped with four gates and a fast PMT (10 MHz) could be slower than a TCSPC at low count-rates (<100 kHz), because of a lower photon-economy. However, already at 1 MHz, the former would be almost three times faster and more the one order of magnitude faster at 10 MHz. 

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. 

Abbreviations BSA, bovine serum albumin PBS, phosphate buffered saline PMFS, phase-modulation fluorescence spectroscopy TCSPC, lime-correlated single photon counting. 

Becker, W., Hickl, H., Zander, C., et al. 1999. Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (Tcspc). Rev. Sci. Instrum. 70 1835. 

Duncan, R.R., Bergmann, A., Cousin, M.A., Apps, D.K., and Shipston, M.J. et al. 2004. Multidimensional time correllated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (Aim) to Detect Fret in Cells. J. Microsc. 215 1. 

Time-Correlated Single-Photon Counting. For the application of TCSPC in the picosecond time domain, lasers with pulses whose half-widths are 20 ps or less are used. For better time resolution, the combination of a microchan-nel plate photomultiplier tube (MCP-PMT) and a fast constant fraction discriminator (CFD) are used instead of a conventional photomultiplier tube (PMT). A TCSPC system with a time response as short as 40 ps has at its core a Nd YLF (neodymium yttrium lithium fluoride) laser generating 70-ps, 1053-nm pulses at... 

Time-correlated single photon counting (TCSPC) [28] is one of the most sensitive methods for studying time-resolved emission. In this technique, single photon events are detected after excitation and a statistical distribution of photons representing the decay of the excited state is built up over time. 

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 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. 

Time-resolved PL measurements were also performed using time-correlated single-photon counting (TCSPC) and photoluminescence upconversion (PLUC) spectroscopies. Descriptions of the setups can be found in refs. [14, 65], respectively. All measurements were taken in continuous-flow He cryostats (Oxford Instruments OptistatCF) under inert conditions. Finally, PL efficiency measurements were performed on simple polymer thin films spin coated on Spectrosil substrates using an integrating sphere coupled to an Oriel InstaSpec IV spectrograph and excitation with the same Ar+ laser as above. 

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