Fluorescence time-delayed

We have implemented the discrimination in the frequency domain. As is known in multifrequency phase fluorometry,17 the time-delayed fluorescence acquires a phase shift >p and a reduction in amplitude Mp upon increasing the modulation frequency m = 2irf of the sinusoidally modulated excitation. For a simple single exponential decay, this phase shift

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Phosphorescence can be observed without interference from fluorescence by a process called time resolution. Instruments for measuring phosphorescence are very similar to those used for fluorescence but a mechanism that allows the sample to be irradiated and then, after a time delay, allows measurement of phosphorescent intensity (phosphoroscope) is required as an extra component. The instrument should also have the capability of keeping samples at very low temperatures. Another type of long-lived photoluminescence is time-delayed fluorescence, where the electrons in the molecule obtain enough energy to be excited from a special excited state to the normal excited state and then fluoresce. 

Keywords Autofluorescence Decay time Delayed fluorescence Fluorescence lifetime imaging microscopy Lanthanides Phosphorescence Time-correlated single photon counting Time-resolved fluorescence microscopy... 

Figure Al.6,8 shows the experimental results of Scherer et al of excitation of I2 using pairs of phase locked pulses. By the use of heterodyne detection, those authors were able to measure just the mterference contribution to the total excited-state fluorescence (i.e. the difference in excited-state population from the two units of population which would be prepared if there were no interference). The basic qualitative dependence on time delay and phase is the same as that predicted by the hannonic model significant interference is observed only at multiples of the excited-state vibrational frequency, and the relative phase of the two pulses detennines whether that interference is constructive or destructive.

CN] —> I + CN. Wavepacket moves and spreads in time, with its centre evolving about 5 A in 200 fs. Wavepacket dynamics refers to motion on the intennediate potential energy surface B. Reprinted from Williams S O and lime D G 1988 J. Phys. Chem.. 92 6648. (c) Calculated FTS signal (total fluorescence from state C) as a fiinction of the time delay between the first excitation pulse (A B) and the second excitation pulse (B -> C). Reprinted from Williams S O and Imre D G, as above. 

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... 

Figure 9.43 The effect on time delay measurement of fluorescence intensity from sodium atoms in a transition state of Nal of changing the pump wavelength to (a) 300 nm, (b) 311 nm, (c) 321 nm, and (d) 339 nm. (Reproduced, with permission, from Rose, T. S., Rosker, M. J. and Zewail, A. H., J Chem. Phys., 91, 7415, 1989)...

Figure 1.5. Femtosecond spectroscopy of bimolecular collisions. The cartoon shown in (a illustrates how pump and probe pulses initiate and monitor the progress of H + COj->[HO. .. CO]->OH + CO collisions. The huild-up of OH product is recorded via the intensity of fluorescence excited hy the prohe laser as a function of pump-prohe time delay, as presented in (h). Potential energy curves governing the collision between excited Na atoms and Hj are given in (c) these show how the Na + H collision can proceed along two possible exit channels, leading either to formation of NaH + H or to Na + H by collisional energy exchange.

Homogeneous Time Resolved Fluorescence (HTRF) (Cisbio International) is an assay based on the proximity of a lanthanide cryptate donor and a fluorescent acceptor molecule whose excitation wavelength overlaps that of the cryptate s emission. The utility of this technique is based on the time resolved fluorescence properties of lanthanides. Lanthanides are unique in the increased lifetime of their fluorescence decay relative to other atoms, so a delay in collection of the emission intensity removes the background from other fluorescent molecules. An example of the HTRF assay is a generic protein-protein interaction assay shown in Fig. 2. 

Many compounds are capable of absorbing uv radiation and subsequently emitting radiation of a longer wavelength, either instantly (fluorescence) or after a time delay (phosphorescence). Usually, the fraction of the absorbed energy that is re-emitted is quite low, but for a few compounds values of 0.1-1 are obtained, and such compounds... 

Marriott, G., Clegg, R. M., Arndt-Jovin, D. J. and Jovin, T. M. (1991). Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging. Biophys. J. 60, 1374—87. 

Flash photolysis studies with absorption or delayed fluorescence detection were performed to compare the binding of ground and excited state guests with DNA.113,136 The triplet lifetimes for 5 and 6 were shown to be lengthened in the presence of DNA.136 The decays were mono-exponential with the exception of the high excitation flux conditions where the triplet-triplet annihilation process, a bimo-lecular reaction, contributed to the decay. The residence time for the excited guest was estimated to be shorter than for the ground state, but no precise values for the rate constants were reported. However, the estimated equilibrium constants for the... 

Also there seems to be a certain time delay between photoreaction and complete recovery of the nematic phase. This problem is relevant to molecular mobility in liquid crystals as a function of temperature, rubbing condition, external electric field and most importantly, the type of liquid crystal. Research is now being undertaken on direct determination of molecular mobility by fluorescence technique. 

By varying the time delay between the pump and probe pulses, the resulting fluorescence brought about by the 388 nm pulse can be determined as shown in Figure 10.15. ... 

Triplet-triplet annihilation In concentrated solutions, a collision between two molecules in the Ti state can provide enough energy to allow one of them to return to the Si state. Such a triplet-triplet annihilation thus leads to a delayed fluorescence emission (also called delayed fluorescence of P-type because it was observed for the first time with pyrene). The decay time constant of the delayed fluorescence process is half the lifetime of the triplet state in dilute solution, and the intensity has a characteristic quadratic dependence with excitation light intensity. 

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