Fluorescence spectroscopy time-correlated single-photon counting

D. J. S. Birch and R. E. Imhof, Time-domain fluorescence using time correlated single-photon counting, in Topics in Fluorescence Spectroscopy (J. R. Lakowicz, ed.), Vol 1, pp 1-95, Plenum Press, New York (1991). 

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. 

The rotational reorientation times of the sample in several solvents at room temperature were measured by picosecond time-resolved fluorescence and absorption depolarization spectroscopy. Details of our experimental setups were described elsewhere. For the time-correlated single photon counting measurement of which the response time is a ut 40 ps, the sample solution was excited with a second harmonics of a femtosecond Ti sapphire laser (370 nm) and the fluorescence polarized parallel and perpendicular to the direction of the excitation pulse polarization as well as the magic angle one were monitored. The second harmonics of the rhodamine-640 dye laser (313 nm 10 ps FWHM) was used to raesisure the polarized transient absorption spectra. The synthesis of the sample is given elsewhere. All the solvents of spectro-grade were used without further purification. 

Steady-state and time-resolved fluorescence spectroscopy Absorption and fluorescence spectra were measured with a Hitachi 557 spectrophotometer and a Hitachi 850 spectrofluorometer, respectively. The time-resolved fluorescence spectra were measured with the apparatus reported previously [4,6] in principle, the time-correlated single photon counting system under a low excitation condition. The pulse intensity (540 nm, 6 ps (fwhm)) was in a range of 10 to 10 photons/cm. The time resolution of our optical set-up was 6 ps. Correction of spectral sensitivity and data treatment were carried out as reported previously [4,6]. 

FCS fluorescence correlation spectroscopy PCH photon counting histogram TCSPC time correlated single photon counting MCS muiti-channei scaiar APDiavaianche photodiode PMT photo-mutipiiertube PCi peripherai component interconnect. 

Harris describe a method for the quantitative estimation of component amplitudes in multiexponential data obtained from time-resolved fluorescence spectroscopy. A design of apparatus which uses time correlated and single photon counting with alternate recording of excitation and emission minimises troublesome lamp... 

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