Fluorescence decay method

H-NMR spectra of the procyanidins, like most other proanthocyanidins, show restricted rotation about the interflavanoid bond under normal temperature conditions (91, 98, 352, 382). The phenolic forms of procyanidins with a 2,3-cis-3A-trans upper unit give broadened but first order spectra until the temperature is reduced to 0 C where two rotational isomers become apparent (98). It is very important to establish the presence and relative proportions of rotational isomers in the free phenols at physiological temperature conditions. It is not possible to resolve these rotamers by NMR because of the comparatively slow time scale. The presence of two rotamers of the dimeric procyanidins as free phenols, and in proportions similar to those found for the locked methyl ether or acetate derivatives, has recently been shown by time-resolved fluorescence decay methods... 

For fluorescent compounds and for times in die range of a tenth of a nanosecond to a hundred microseconds, two very successftd teclmiques have been used. One is die phase-shift teclmique. In this method the fluorescence is excited by light whose intensity is modulated sinusoidally at a frequency / chosen so its period is not too different from die expected lifetime. The fluorescent light is then also modulated at the same frequency but with a time delay. If the fluorescence decays exponentially, its phase is shifted by an angle A([) which is related to the mean life, i, of the excited state. The relationship is... 

There are, however, problems associated with this method of determining fluorescence lifetimes. First, the phase method is not generally applicable for nonexponential signals and, as we shall see later, there are many cases where the observed fluorescence decay is indeed nonexponential. Second, the method... 

A linear plot indicates that the luminescence decay is exponential. The slope of the line gives kt, and rt can be calculated as above. The lifetime obtained by measuring the decay of P-type delayed fluorescence is equal to one-half the lifetime of the triplet state (see Section 5.2). Since in fluid solution at room temperature phosphorescence is generally much weaker than delayed fluorescence, the measurement of delayed fluorescence decay offers a convenient method for determining the lifetime of triplets at room temperature. 

FRET applications employing CFP and YFP are complicated due to considerable bleed-through between CFP and YFP fluorescence (Figs. 5.5B and 5.6B). Direct excitation of YFP and bleed-through of CFP fluorescence into the YFP detection channel have to be corrected for as shown in Chapters 7 and 8. The multiexponential fluorescence decay of all CFP variants complicates the quantification of FRET by donor lifetime methods. Altogether these factors make quantitative analysis of the FRET efficiency relatively difficult. 

Optical sensors for oxygen measurement are attractive since they can be fast, do not consume oxygen and are not easily poisoned. The most common method adopted in construction is based on quenching of fluorescence from appropriate chemical species. The variation in fluorescence signal (I), or fluorescence decay time (x) with oxygen concentration [O2] is described by Stem-Volmer equation91 ... 

To answer the question as to whether the fluorescence decay consists of a few distinct exponentials or should be interpreted in terms of a continuous distribution, it is advantageous to use an approach without a priori assumption of the shape of the distribution. In particular, the maximum entropy method (MEM) is capable of handling both continuous and discrete lifetime distributions in a single analysis of data obtained from pulse fluorometry or phase-modulation fluorometry (Brochon, 1994) (see Box 6.1). 

Eaton D. F. (1990) Recommended Methods for Fluorescence Decay Analysis, Pure Appl. Chem. 62, 1631-1648. 

Time-resolved method 1 decay of the donor fluorescence If the fluorescence decay of the donor following pulse excitation is a single exponential, the measurement of the decay time in the presence (td) and absence (t ) of transfer is a straightforward method of determining the transfer rate constant, the transfer efficiency and the donor-acceptor distance, by using the following relations ... 

The advantage of this method is its ability to check whether the donor fluorescence decay in the absence and presence of acceptor is a single exponential or not. If this decay is not a single exponential in the absence of acceptor, this is likely to be due to some heterogeneity of the microenvironment of the donor. It can then be empirically modeled as a sum of exponentials ... 

Intramolecular charge transfer in p-anthracene-(CH2)3-p-Ar,Af-dimethylaniline (61) has been observed174 in non-polar solvents. Measurements of fluorescence-decay (by the picosecond laser method) allow some conclusions about charge-transfer dynamics in solution internal rotation is required to reach a favourable geometry for the formation of intramolecular charge-transfer between the donor (aniline) and the acceptor (anthracene). 

Some methods of quantitative analysis of nonexponential fluorescence decay curves will be shortly described in Section 6.6. 

In this section, the information on structure and dynamics of proteins which may be obtained from direct observations of fluorescence decay will be considered. This type of information is afforded by methods which permit fluorescence decay kinetics to be followed with picosecond and nanosecond resolution. 

The polarization properties of light in combination with fluorescence can be used as a powerful tool for determining motional properties of membranes. This is possible due to the fact that the time scale of interest for membrane lipids falls within the time frame of the fluorescence decay phenomena (0-100+ ns). This, coupled with high sensitivity, low perturbing properties of fluorescent probes, and the large number of available probes, makes the fluorescence approach the method of choice for membrane motional studies. 

Selected entries from Methods in Enzymology [vol, page(s)] Analysis of GTP-binding/GTPase cycle of G protein, 237, 411-412 applications, 240, 216-217, 247 246, 301-302 [diffusion rates, 246, 303 distance of closest approach, 246, 303 DNA (Holliday junctions, 246, 325-326 hybridization, 246, 324 structure, 246, 322-324) dye development, 246, 303, 328 reaction kinetics, 246, 18, 302-303, 322] computer programs for testing, 240, 243-247 conformational distribution determination, 240, 247-253 decay evaluation [donor fluorescence decay, 240, 230-234, 249-250, 252 exponential approximation of exact theoretical decay, 240, 222-229 linked systems, 240, 234-237, 249-253 randomly distributed fluorophores, 240, 237-243] diffusion coefficient determination, 240, 248, 250-251 diffusion-enhanced FRET, 246, 326-328 distance measurement [accuracy, 246, 330 effect of dye orientation, 246, 305, 312-313 limitations, 246,... 

Selected entries from Methods in Enzymology [vol, page(s)[ Analysis, 240, 290-310 anisotropic, 240, 301-310 effect of material diffusion, 240, 219, 221 evaluation of donor fluorescence decay, 240, 230-234 optimal length of time step, 240, 224-229 exponential approximation of exact theoretical decay, 240, 222-229 linked systems, 240, 234-237 measurement techniques... 

In kinetic analysis of complex reactions, 210, 382 fluorescence decay rate distributions, 210, 357 implementation in Laplace de-convolution noniterative method, 210, 293 in multiexponential decays, 210, 296 partial global analysis by simulated annealing methods, 210, 365 spectral resolution, 210, 299. 

There have been major advances in the development of both methods in recent years and fluorescence lifetime measurements on the picosecond time scale are now quite feasible. Precise analysis of the fluorescence decay revealed that even in the case of a peptide containing a single fluorophore multiexponential decay rather than single-exponential decay is often observed, as described by eq 5... 

Conventional EPR techniques have been successfully used to measure the D and E values of matrix-isolated carbenes in the ground triplet state because the steady-state concentration of triplet species is sufficiently high in the system. The technique cannot be used, however, for excited species having triplet hfetimes of the order of 10-100 ns, since their steady-state concentration is too low. The D parameters are estimated from the external magnetic field effect on the T—T fluorescence decay in a hydrocarbon matrix at low temperamre. The method is based on the effect of the Zeeman mixing on the radiative and nonradiative decay rates of the T -Tq transition in the presence of a weak field. The D values are estimated by fitting the decay curve with that calculated for different D values. The D T ) values estimated for nonplanar DPC (ci symmetry) is 0.20... 

Glasses. Pearson and Peterson 98) studied extensively the fluorescent decay properties of terbium in Calibo base glass which has the composition CaO, 20 Li20, 10 B203, 70 mole per cent. All their data was taken using a stroboscopic method. 

Rieke and Allison (97) also examined the fluorescent-decay time of europium dibenzoylmethide (EuD3). They found a marked difference between it and unchelated europium compounds. The material was prepared by the method described by Crosby et al. (142). The data were collected by illuminating the samples with a stroboscopic light source of 20-/zsec decay time. The results for EuD3 as compared with their data on EuC13-4H20 are ... 

Metlay (145) studied the fluorescent decay of both EuD3 and EuD4. These compounds were prepared by the method of Crosby et al. (142). As Metlay points out, Whan and Crosby (146) in a later paper describe their preparation in more detail. They indicate that a period of heating in vacuum is necessary to convert the chelate from one containing four molecules of dibenzoylmethane per atom of europium to one containing but three. 

Abstract Ultrafast photoreactions in PNS of PYP have been studied by means of fs fluorescence up conversion method. Conclusions obtained are (a) Photoreaction in PNS (chromophore twisting) occurs from vibrationally unrelaxed fluorescence state and coherent oscillations in the fluorescence decay curves have been observed for the first time, (b) Comparative studies on fluorescence dynamics of mutants and w.-t. PYP have proved that the w.-t. PYP is best engineered for the ultrafast reaction, (c) The coherent oscillations in the fluorescence decay completely disappeared and the reaction was much slower in the denatured state, demonstrating the supremely important role of PNS for the photoreaction. 

Instruments of this type may also be used quite effectively to evaluate kinetics of time-dependent changes in foods, be they enzymatic or reactive changes of other types. The computerized data-acquisition capabilities of these instruments allow precise measurement of absorbance or fluorescence changes, often over very brief time periods ( milliseconds). This is particularly useful for analysis of fluorescence decay rates, and in measurement of enzymatic activity in situ. A number of enzyme substrates is available commercially which, although non-fluorescent initially, release fluorescent reaction products after hydrolysis by appropriate enzymes. This kinetic approach is a relatively underused capability of computerized microspectrophotometers, but one which has considerable capability for comparing activities in individual cells or cellular components. Fluorescein diacetate, for example, is a non-fluorescent compound which releases intensely fluorescent fluorescein on hydrolysis. This product is readily quantified in individual cells which have high levels of esterase [50]. Changes in surface or internal color of foods may also be evaluated over time by these methods. 

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