Time-Correlated Single Photon Counting TCSPC

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. 

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 (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... 

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. 

Time-Correlated Single Photon Counting (TCSPC)... 

While the above-described instrumentation is an excellent choice for long-lived excited states such as the Ru " and Os polypyridyl complexes, organic-based chromophores and fluorescence labels frequently used by supramolecular chemists require higher time resolution. Commercially available time-correlated single photon counting (TCSPC) instruments can readily access... 

Luminescence lifetimes measured using pulsed laser excitation involve either direct detection of emission decays with time or a technique known as time-correlated single photon counting (TCSPC). The latter technique involves repeated measurement of the delay time between the excitation pulse and the arrival of an emitted photon packet above a given discrimination level the intensity-time decay profile accumulates over many millions of excitation pulses. The TCSPC experiment has the advantage that much better signal to noise can be obtained relative to the direct capture of the luminescence decay. 

The lifetime of the first excited state of single pentacene molecules in p-terphenyl was measured by time correlated single photon counting (TCSPC) [10]. The expected lifetime of the 5 -state of pentacene is about 20 ns, and it was necessary to optimize the excitation laser pulse duration carefully. While short pulses are advantageous. 

To obtain fluorescence lifetimes time correlated single photon counting (TCSPC) was used. In TCSPC, the elapsed time is measured between an excitation pulse from a pulsed laser and a detected photon. A histogram of the elapsed times provides a fluorescence decay curve, from which the fluorescence lifetime, Zf, is extracted. Examples of decay curves for bare silica and single R6G molecules on silica taken with a near-field probe are shown in Fig. 11 [21]. 

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