Measurement of fluorescence lifetime

The validity of the above conclusions rests on the reliability of theoretical predictions on excited state barriers as low as 1-2 kcal mol . Of course, this required as accurate an experimental check as possible with reference to both the solvent viscosity effects, completely disregarded by theory, and the dielectric solvent effects. As for the photoisomerization dynamics, the needed information was derived from measurements of fluorescence lifetimes (x) and quantum yields (dielectric constant, where extensive formation of ion pairs may occur [60], the observed photophysical properties are confidently referable to the unperturbed BMPC cation. Figure 6 shows the temperature dependence of the... 

At present, two main streams of techniques exist for the measurement of fluorescence lifetimes, time domain based methods, and frequency domain methods. In the frequency domain, the fluorescence lifetime is derived from the phase shift and demodulation of the fluorescent light with respect to the phase and the modulation depth of a modulated excitation source. Measurements in the time domain are generally performed by recording the fluorescence intensity decay after exciting the specimen with a short excitation pulse. 

Hedstrom, J., Sedarous, S. and Prendergast, F. G. (1988). Measurements of fluorescence lifetimes by use of a hybrid time-correlated and multifrequency phase fluorometer. Biochemistry 27, 6203-8. 

Figure 11.9. Phase and modulation measurement of fluorescence lifetime, tan = 2 jr/r = phase difference.

The measurement of fluorescence lifetimes is an integral part of the anisotropy, energy transfer, and quenching experiment. Also, the fluorescence lifetime provides potentially useful information on the fluorophore environment and therefore provides useful information on membrane properties. An example is the investigation of lateral phase separations. Recently, interest in the fluorescence lifetime itself has increased due to the introduction of the lifetime distribution model as an alternative to the discrete multiexponential approach which has been prevalent in the past. 

It should be noted that the existence of different centers Is also found in covalently-linked dimer 11. Moreover, the analysis of all data obtained for dimers points clearly towards the efficient transfer of the excited singlet state energy from two centers of compound 1 to two acceptor centers of compound 2 in dimers (14,30). Increase In the porphyrin concentration by 300-700 times (say, for compounds 1 and 2) does not cause additional changes In electronic spectra as against diluted solutions. If the results obtained from temperature experiments (Fig. 2b) and measurements of fluorescence lifetimes In different bands are taken Into account, one may conclude that the additional centers observed In... 

Overview. The proposed near-term research is divided into three areas, which will be pursued simultaneously. The first is the complete characterization of the current mercury-responsive fluorescent chemosensor system, including the measurement of fluorescence lifetimes, to discern the origin of the conformational control of fluorescence. The second is to develop two classes of substituted biaryl acetylenic fluorescent chemosensors, to move the observed fluorescence into the visible region and increase the magnitude of the fluorescence signal, which occurs upon conformational restriction. 

A simplified instrument for the measurement of fluorescent lifetimes using the stroboscopic method has been described by Brown (67). The major virtue of this system is that it makes use of a Tektronix oscilloscope to obtain all the necessary trigger pulses, including a trigger of continuously variable delay. Since most laboratories are equipped with a good oscilloscope, the need to purchase expensive trigger-delay apparatus is thus eliminated. 

Phase sensitivity A phase-sensitive flow cytometer quantifies the life time of the fluorescence emitted by particles. The decay time of fluorescence from a given fluorochrome is altered by changing chemical environments, and therefore measurement of fluorescence lifetimes can provide information about the microenvironment surrounding the fluorescent probe. 

Fig. 8. Block diagram of a typical single-photon counting apparatus for the measurement of fluorescence lifetimes.

As a summary to this section dealing with the measurement of fluorescence lifetimes, Table 1 gives some examples of lifetime determinations for some diatomic molecules. The majority of the studies cited in Table 1 have been made since 1975. The quantity of data available, even just for diatomic molecules, is large and it is impossible to include all lifetime measurements in such a brief table. However, the examples in Table 1 do give some impression of the wide range of species to which the techniques discussed above can, and have, been applied. 

Laser-Induced Fluorescence. Laser-induced fluorescence (Lif) provides, much as does ir spectroscopy, fingerprints of different organic molecules, which can be quantified by measuring fluorescence intensities. Selectivity is excellent, as both pump and fluorescence frequencies can be individually chosen for optimum performance, and it can be improved with measurements of fluorescence lifetimes and polarization behavior. The enhanced null-background sensitivity can achieve single-atom or single-molecule detection (256—258). Lif has important applications in gas analysis (259) and combustion and plasma diagnostics (260). 

Over a substantial number of years the phase-shift or frequency-domain method has been employed for the measurement of fluorescence lifetimes. The technique requires the continuous excitation of a fluorescent sample with a source of varying intensity. The fluorescence response would normally be expected to increase and decrease to reflect the changes in excitation intensity. However, in a frequency-domain experiment the excitation beam is modulated at a high frequency, (o = 2nf, to produce a sinusoidally changing intensity given by ... 

Measurements of fluorescence lifetime ofa chromophore can enhance the potential offluorescence microscopy [1,2,5-8]. Fluorescence lifetime is an inherent property of a chromophore, and thus is independent of chromophore concentration, photo-bleaching and, excitation intensity, but highly dependent on pH, ion concentration, and local environment that affects the non-radiative rate of a chromophore. This makes fluorescence lifetime imaging (FLIM) a powerful tool for quantitative imaging of cellular conditions as well as the circumstances around the fluorescent dyes. 

Spectroscopists have shown many powerful variations on the fluorescence experiment, which can generate additional selectivity and structural information. These include two-photon excitation (55) supersonic jet expansion (56) constant-energy synchronous fluorescence, where the excitation and emission monochromators are scanned synchronously to maintain a constant energy difference (57) and pulsed excitation for the real-time measurement of fluorescence lifetimes (58). However, it is far from certain that these techniques will ever be useful for the practicing analyst. 

R.D. Spencer, G. Weber, Influence of brownian rotations and energy transfer upon the measurement of fluorescence lifetime, J. Chem. Phys. 52 1654-1663 (1970)... 

Laser Exdtation.—Introduction. With the advent of narrow bandwidth, pulsed, tunable dye lasers within the past five years, direct measurements of fluorescence lifetimes and quendiing cross-sections, as a function of individual vibration-rotation states in electronically excited states, have become available. The nanosecond pulses available from variously pumped dye lasers have proved particularly useful for measurements of lifetimes of excited states, typically of the order of 0.01—100/is. 

Some information on the microviscosity can also be obtained by steady-state anisotropy measurements. A comparison of results for different media (e.g., for a series of mixed solvents differing in composition) is tricky and requires the measurement of fluorescence lifetimes. The steady-state anisotropy, (r), is a time-average weighted by the fluorescence intensity decay, I t) ... 

In our discussion of instrumentation foctors, we will stress their effects on excitation and emission spectra. However, similar concerns are inqx>rtant in the measurement of fluorescence lifetimes and anisotropies, which will be described in Chapters 4, 5, and 10. Additionally, the optical prc rties of the samples, such as optical density and turbidity, can also affect the spectra. Specific examples are given to clarify these effects and the means to avoid them. 

Previously, we have studied fluorescence quenching in unsolvated dye-derivatized biomolecules by performing measurements of fluorescence lifetime, intensity and emission spectrum as a function of temperature. These studies have identified that fluorescence quenching is dependent on conformational dynamics which lead to interactions between the dye and a Trp (Tryptophan) side chain in peptides [34,35] and small protein [36,37] structures. 

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