Fluorescence pulse fluorimetry

Criteria for evaluating the degree of fit between measured fluorescence decay curves and trial decay functions have been discussed.In some instances plots of weighted residuals were found to be sufficient, but a generalized statistical test was proposed for all other cases. An analysis of the statistical distribution of noise in fluorescence decay measurements by SPC has shown, as expected, that Poisson statistics dominate. A method for obtaining decay information from pulse fluorimetry without the need for consideration of the excitation pulse, has been described. ... 

Feenqr, J., Fluorescence Nanosecond Pulse Fluorimetry, in New Techniques in Biophysics CeVLBicAogy, Vol. 2, pp. 233-287, (R.H. Pain and B.J. Smith, eds.), Jolm Wiley, London. 1975. 

Both TCSPC and frequency-domain fluorimetry are limited in time resolution by the response of available detectors, typically >25 ps. For cases in which higher time resolution is needed, fluorescence up-conversion can be used (22). This technique uses short laser pulses (usually sub-picosecond) both to excite the sample and to resolve the fluorescence decay. Fluorescence collected from the sample is directed through a material with nonlinear optical properties. A portion of the laser pulse is used to gate the fluorescence by sum frequency generation. The fluorescence is up-converted to the sum frequency only when the gate pulse is present in the nonlinear material. The up-converted signal is detected. The resolution of the experiment therefore depends only on the laser pulse widths and not on the response time of the detectors. As a result, fluorescence can be resolved on the 100-fs time scale. For a recent application of fluorescence up-conversion to proteins, see Reference 23. 

Time-resolved fluorimetry is also useful for the elimination of interferences from stray light due to Rayleigh and Raman scatter. The latter phenomena occur on a time scale of l(r14-l(T13 s and, as they have a much shorter duration than lamp or laser pulses, the light associated with them can be eliminated from the signal that ultimately reaches the detector. Time-correlated single-photon counting is superior in its ability to resolve multiple fluorescence from the same solution. 

By solving the inverse problem, the fluorescence lifetime r of a fluorophore can be determined independently from the dependence Nn(tdei). In the experiment the fluorescenoe signal is measured in relative units. For comparison of the experimental data with the theoretical ones it is necessary to normalize the obtained experimental curve to the fluorescence intensity at some fixed time delay. This procedure, the fluorimeter capabiUties in the kinetic mode and the corresponding theory can be found elsewhere (Banishev et al., 2006). The difference of such variant of kinetic fluorimetry from the conventional time-resolved fluorimetiy is that the fluorescence is excited by a pulse with rather long duration ( 10 ns), and for fluorescence registration an optical gated multichannel analyser is used. Whereas, the conventional time-resolved fluorimetry (Lakowicz, 1999) is based on the analysis of fluorescence decay curves after the excitation pnilse, whose duration is much shorter than the lifetime of a fluorophore in the excitation state (picoseconds). 

Time-resolved fluorescence measurements are performed in two functionally equivalent ways time domain and frequency domain. In time-domain or pulse-excitation fluorimetry, the fluorophore is excited with a short-duration pulse of light. The resulting fluorescence emission decay signal after the excitation pulse is captured with a fast recorder and the decay time is calculated from the slope of a plot of log I(t) versus t. As the fluorescence species decay involves a process that occurs in a few nanoseconds. 

Two time-resolved fluorescence techniques, pulse Jluorimetry and phase-modulation fluorimetjy, are commonly employed to recover the lifetimes. The former uses a short exciting pulse (from femtoseconds to nanoseconds) of light, which leads to the pulsed response of the sample, which should then be deconvolved from the instrument response. In phase-modulation fluorimetry, the intensity of light used for excitation is modulated at a frequency whose reciprocal is similar to the fluorescence decay time. The sample response is also modulated, but with a time delay, measured as phase shift, from which the emission decay time can be calculated. Thus, the first technique works in the time domain, while the second one in the frequency domain. The most widely used technique in the time domain is the time-correlated single-photon counting [10, 11]. The merits of both techniques have been extensively discussed [12]. 

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