Fluorometry, time-resolved

Knowledge of the dynamics of excited states is of major importance in understanding photophysical, photochemical and photobiological processes. Two time-resolved techniques, pulse fluorometry and phase-modulation fluorometry, are commonly used to recover the lifetimes, or more generally the parameters characterizing the S-pulse response of a fluorescent sample (i.e. the response to an infinitely short pulse of light expressed as the Dirac function S). 

Pulse fluorometry uses a short exciting pulse of light and gives the d-pulse response of the sample, convoluted by the instrument response. Phase-modulation fluorometry uses modulated light at variable frequency and gives the harmonic response of the sample, which is the Fourier transform of the d-pulse response. The first technique works in the time domain, and the second in the frequency domain. Pulse fluorometry and phase-modulation fluorometry are theoretically equivalent, but the principles of the instruments are different. Each technique will now be presented and then compared. 

In some cases, filters are used instead of the emission monochromator. In principle, no G factor is then considered, but in practice, effects may be due to the sensitivity of the photomultipliers to polarization (in particular, photomultipliers with side-on photocathodes). 

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C. Single-Molecule Imaging and Time-Resolved Fluorometry of TNP-ATP... 

This class of ligands mostly consists of derivatives of pyridine, 2.2 -bipyridinc. 2,2, 2"-ter-pyridine, and 1,10-phenanthroline. Scheme 6 shows the structures of fourEu3+ chelates with aromatic amine derived ligands, that can be covalently bound to proteins, for time-resolved fluorometry, among which 4,7-bis(chlorosulfophenyl)-l,10-phenanthroline-2,9-dicarboxylic acid (BCPDA)-Eu3+ and trisbipyridine cryptate (TBP)-Eu3+ are widely used for europium fluorescence labels in TR-FIA. 

The emission-decay profiles of Tb3+, Dy3+, Eu3+ and Sm3+ complexes of pivaloyl trifluoroacetone are shown in Fig. 12.31. A sketch of time resolved fluorometry of Eu3+ with spectral filters and temporal resolution (time windows) is shown in Fig. 12.32. 

Fig. 12.32. Time-resolved fluorometry of Eu3+ with combination of spectral (filters) and temporal (time...

The decay times and detection limits of some lanthanides measured with time resolved fluorometry using different enhancement systems are given in Table 12.33. 

Besides improving classical electrochemical methods, newly employed techniques such as second harmonic-generation and time-resolved fluorometry, with either control of the potential drop across the interface or fluctuation analysis, are promising in this respect. Also indispensable are further advances in molecular dynamics and statistical-mechanical treatments of structure and charge transfer at the ITIES. 

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