Lifetimes single-photon counting

The other coimnon way of measuring nanosecond lifetimes is the time-correlated single-photon counting... 

Figure C1.5.12.(A) Fluorescence decay of a single molecule of cresyl violet on an indium tin oxide (ITO) surface measured by time-correlated single photon counting. The solid line is tire fitted decay, a single exponential of 480 5 ps convolved witli tire instmment response function of 160 ps fwiim. The decay, which is considerably faster tlian tire natural fluorescence lifetime of cresyl violet, is due to electron transfer from tire excited cresyl violet (D ) to tire conduction band or energetically accessible surface electronic states of ITO. (B) Distribution of lifetimes for 40 different single molecules showing a broad distribution of electron transfer rates. Reprinted witli pennission from Lu andXie [1381. Copyright 1997 American Chemical Society.

The lifetime resolution is the smallest variation in lifetime that can be detected. If external noise sources are ignored, the lifetime resolution depends essentially on the photon-economy of the system. For instance, if a 2 ns lifetime is measured with a 4 gate TG single-photon counting FLIM (F = 1.3) and 1000 photons, variations of about 80 ps can be resolved. However, for reasons discussed earlier, in biological samples these values could be higher. 

Becker, W., Bergmann, A., Hink, M. A., Konig, K., Benndorf, K. and Biskup, C. (2004b). Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc. Res. Tech. 63, 58-66. 

For fluorescence decay curves of the J-aggregate LB films of [CI-MC] mixed with various matrix agents, measured with a picosecond time-resolved single photon counting system, three components of the the lifetimes fitting to exponential terms in the following equation ... 

The technique of time-correlated single-photon counting (Figure 3.4) is used to measure an excited singlet-state lifetime, x. The sample is irradiated with a very short-duration light pulse ( lns) to ensure any given molecule will only be excited once during the pulse. As soon as... 

H. E. Zimmerman, J. H. Penn, and C. W. Carpenter, Evaluation of single-photon counting measurements of excited-state lifetimes, Proc. Natl. Acad. Sci. USA 79,2128-2132 (1982). 

The potential for using multiplexed single-photon counting to acquire fluorescence lifetime data from a distributed array of optical fiber sensors, each sensing a different analyte, has recently been demonstrated by Birch etal.(39,47) Figure 12.9 illustrates such a network to be used in conjunction with the arrangement shown in Figure 12.6. 

T. A. Louis, G. Ripamonti and A. Lacaita, Photoluminescence lifetime microscope spectrometer based on time-correlated single-photon counting with an avalanche diode detector, Rev. Sci Instrum. 61, 11-22(1990). 

K. J. Willis, A. G. Szabo, and D. T. Krajcarski, The use of Stokes Raman scattering in time correlated single photon counting Application to the fluorescence lifetime of tyrosinate,... 

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. 

Instrumentation. The steady-state fluorescence spectra were measured with Perkin-Elmer MPF-44B fluorescence spectrophotometer. The single-photon counting instrument for fluorescence lifetime measurements was assembled in-house from components obtained from EG G ORTEC. A PRA-510B light pulser filled with gas was used as the excitation source. Instrument response function was obtained with DuPont Ludox scatter solution at the excitation wavelength. 

The fluorescence of 2AP is strongly quenched by nucleic acid bases [17, 18, 24-29]. Time-correlated single-photon counting studies have shown that the interactions of 2AP with different nucleic acid bases significantly decrease the 2AP fluorescence hfetime [17, 24-29]. While the fluorescence lifetime of free 2AP in aqueous solution is about 10 ns, in double-stranded DNA the 2AP hfetimes are reduced to 30-50 ps. This effect has been used extensively to study the dynamics of mismatched base pairs [19, 21, 25, 30], local changes in dynamics of DNA molecules produced by their binding to the active sites of polymerases [26, 31-33], stacking interactions at abasic... 

Nemzek and Ware [7] have studied the fluorescence decay of 1,2-benzanthracene (and naphthalene) in 1,2-propanediol or purified mineral oil by the single photon counting technique over the temperature range 10—45°C. The fluorescence lifetimes, t0, were measured. In further experiments, which included a heavy atom fluorescence quencher, carbon tetrabromide in concentration [Q] 0.05—0.29 mol dm-3, no longer could the decay be characterised by an exponential with a constant lifetime. However, the decay of fluorescence was well described by an expression of the form... 

The probe molecule pyrene (-10"6 M) was used in time-resolved fluorescence quenching experiments using a single photon counting apparatus, cetylpiridinium chloride (CpyC, 10"3 M) being introduced as a quencher of the pyrene fluorescence[ll-13]. All the experiments were performed at 303K. From these fluorescence studies the micelle aggregation number (N) and the pyrene fluorescence lifetime (x) were obtained [14]. 

The decay of phosphorescence emissions can be observed easily with conventional flash photolysis instruments, since they last between ms and seconds. However, fluorescence lifetimes are of the order of ns and such kinetics can be measured only by laser flash photolysis or by time-resolved single photon counting. 

Figure 8.9 Time-resolved fluorescent lifetime analysis of Cy3 attached to double-stranded DNA (Iqbal et al., 2008b). Fluorescent decay curve for Cy3 attached to a 16 bp DNA duplex, showing the experimental data and the instrument response function (IRF), and the fit to three exponential functions (line). The decay curve was generated using time-correlated single-photon counting, after excitation by 200 fs pulses from a titanium sapphire laser at 4.7 MHz.

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