Resonance energy transfer donor lifetimes

If the energy is transferred by trivial emission/reabsorption, it will lengthen the measured lifetime of the donor emission, not shorten it as happens in resonance energy transfer. This comes about because intervening absorption and emission processes take place prior to the final fluorescence emission (the reabsorption cannot take place until the photon has been emitted) the two processes do not compete dynamically, but follow in a serial fashion. In FRET, such an emission/reabsorption process does not occur, and the fluorescence lifetime of the donor decreases. This is an experimental check for reabsorption/reemission. 

The lifetime of the excited state of fluorophores may be altered by physical and biochemical properties of its environment. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful analytical tool for the quantitative mapping of fluorescent molecules that reports, for instance, on local ion concentration, pH, and viscosity, the fluorescence lifetime of a donor fluorophore, Forster resonance energy transfer can be also imaged by FLIM. This provides a robust method for mapping protein-protein interactions and for probing the complexity of molecular interaction networks. 

Forster (1968) points out that R0 is independent of donor radiative lifetime it only depends on the quantum efficiency of its emission. Thus, transfer from the donor triplet state is not forbidden. The slow rate of transfer is partially offset by its long lifetime. The importance of Eq. (4.4) is that it allows calculation in terms of experimentally measured quantities. For a large class of donor-acceptor pairs in inert solvents, Forster reports Rg values in the range 50-100 A. On the other hand, for scintillators such as PPO (diphenyl-2,5-oxazole), pT (p-terphenyl), and DPH (diphenyl hexatriene) in the solvents benzene, toluene, and p-xylene, Voltz et al. (1966) have reported Rg values in the range 15-20 A. Whatever the value of R0 is, it is clear that a moderate red shift of the acceptor spectrum with respect to that of the donor is favorable for resonant energy transfer. 

The sensor for the measurement of high levels of CO2 in gas phase was developed, as well90. It was based on fluorescence resonance energy transfer between 0 long-lifetime ruthenium polypyridyl complex and the pH-active disazo dye Sudan III. The donor luminophore and the acceptor dye were both immobilized in a hydrophobic silica sol-gel/ethyl cellulose hybrid matrix. The sensor exhibited a fast and reversible response to carbon dioxide over a wide range of concentrations. 

The Forster resonance energy transfer can be used as a spectroscopic ruler in the range of 10-100 A. The distance between the donor and acceptor molecules should be constant during the donor lifetime, and greater than about 10 A in order to avoid the effect of short-range interactions. The validity of such a spectroscopic ruler has been confirmed by studies on model systems in which the donor and acceptor are separated by well-defined rigid spacers. Several precautions must be taken to ensure correct use of the spectroscopic ruler, which is based on the use of Eqs (9.1) to (9.3) ... 

What mechanisms can be used to create a lifetime-based glucose sensor In our opinion, the mechanism should be fluorescence resonance energy transfer (FRET). The phenomenon of FRET results in transfer of the excitation from a donor fluorophore to an acceptor chromophore, which need not itself be fluorescent. FRET is a through-space interactor which occurs over distances of 20-60 A. 

Fluorescence resonance energy transfer (FRET) is a spectroscopic means of obtaining distance information over a range up to 80 A in solution. It is based on the dipolar coupling between the electronic transition moments of a donor and acceptor fluorophore attached at known positions on the RNA species of interest. It can be applied in ensembles of molecules, either by steady-state fluorescence or by lifetime measurements, but it is also very appropriate for single-molecule studies. In addition to the provision of distance information, recent studies have emphasized the orientation dependence of energy transfer. 

Fluorescence resonance energy transfer (FRET) luminescence occurs when donor phosphor decreases its emission intensity and luminescent lifetime, while acceptor phosphor lights up. As the precondition of FRET, the donor emission and the acceptor absorption require adequate spectra overlaps. The spatial distance of donor-acceptor pair is the second factor. Only within a small range, the energy could be transferred from donors to... 

As long as r > 3R0, the fluorescence decay is close to exponential, the lifetime of the donor fluorescence decreases linearly with increasing concentration of A and fluorescence quenching obeys Stern Volmer kinetics (Section 3.9.8, Equation 3.36). However, the bimolecular rate constants ket of energy transfer derived from the observed quenching of donor fluorescence often exceed the rate constants of diffusion kd calculated by Equation 2.26, because resonance energy transfer does not require close contact between D and A. Finally, when r < 3R0, at high concentrations and low solvent viscosity, the kinetics of donor fluorescence become complicated, but an analysis is possible,109,110 if required. 

The distance between two different fiuorophore molecules can be probed by fluorescence resonance energy transfer (FRET) [308]. The energy transfer rate from the donor to the aeeeptor depends on the sixth power of the distance. FRET becomes noticeable at distanees on the order of a few mn and therefore occurs only if the donor and aeeeptor are physically linked. With FLIM techniques, FRET results are obtained from a single lifetime image of the donor [15, 32, 38, 61, 62, 63, 73, 80, 93, 147, 209, 405, 508]. 

The ability to measure intensities and lifetimes of both donor and acceptor emission with high accuracy and excellent signal-to-background, coupled with the unusually large R s, makes luminescence resonance energy transfer a potentially powerful technique for measuring distances in biological systems. 

Fluorescence lifetimes are also affected by fluorescence resonance energy transfer (FRET). The phenomenon of FRET is non-radiative energy transfer from the fluorescent donor to the acceptor, without emission and reabsorption of photons. The FRET is completely predictable based on the spectral properties of the donor and acceptor (59-60). If the donor and acceptor are at a distance R within the characteristic Fdrster distance (Ro) for energy transfer (optimal for sensing if 0.8 Rq R 1.3Ro), and if the acceptor absorption spectrum changes in response to the analyte, then the lifetime of the donor will change in response to the analyte. Consequently, FRET can be used for sensing a wide variety of analytes (61-66). 

Fluorescence resonance energy transfer (FRET) experiments commonly use the fluorescent spectrum and relaxation times of the Forster donor and acceptor chromophores to find the distances between fluorescent dyes at labeled sites in protein, DNA, RNA, etc. FRET is a type of spectroscopic ruler . The computation uses either experimental quantum yields or relaxation lifetimes to calculate the efficiency of resonance energy transfer Ej. 

Fluorescence lifetime imaging microscopy (FLIM)-based guantitative fluorescence resonance energy transfer (FRET). Direct detection of biomolecular interactions is possible with FRET measurements, where a donor fluorophore transfers the energy to an acceptor fluorophore in case they are close in space. The combination of this technique together with FLIM, based on the decrease in donor fluorophore lifetime that is induced by FRET, has recently enabled the quantitative assessment of the protein-interacting fractions [12]. 

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