Time-resolved fluorescence spectroscopy

All the techniques already developed for photodissociation dynamics can be used here (laser-induced fluorescence, time resolved spectroscopy, multi photon spectroscopy, etc). 

Hydrogen transfer in excited electronic states is being intensively studied with time-resolved spectroscopy. A typical scheme of electronic terms is shown in fig. 46. A vertical optical transition, induced by a picosecond laser pulse, populates the initial well of the excited Si state. The reverse optical transition, observed as the fluorescence band Fj, is accompanied by proton transfer to the second well with lower energy. This transfer is registered as the appearance of another fluorescence band, F2, with a large anti-Stokes shift. The rate constant is inferred from the time dependence of the relative intensities of these bands in dual fluorescence. The experimental data obtained by this method have been reviewed by Barbara et al. [1989]. We only quote the example of hydrogen transfer in the excited state of... 

The following remarkable features of fluorescence may be observed in polar solutions applying the method of time-resolved spectroscopy ... 

Molecular rotors are useful as reporters of their microenvironment, because their fluorescence emission allows to probe TICT formation and solvent interaction. Measurements are possible through steady-state spectroscopy and time-resolved spectroscopy. Three primary effects were identified in Sect. 2, namely, the solvent-dependent reorientation rate, the solvent-dependent quantum yield (which directly links to the reorientation rate), and the solvatochromic shift. Most commonly, molecular rotors exhibit a change in quantum yield as a consequence of nonradia-tive relaxation. Therefore, the fluorophore s quantum yield needs to be determined as accurately as possible. In steady-state spectroscopy, emission intensity can be calibrated with quantum yield standards. Alternatively, relative changes in emission intensity can be used, because the ratio of two intensities is identical to the ratio of the corresponding quantum yields if the fluid optical properties remain constant. For molecular rotors with nonradiative relaxation, the calibrated measurement of the quantum yield allows to approximately compute the rotational relaxation rate kor from the measured quantum yield [Pg.284]

Lossau H, Rummer A, Heinecke R, Pollinger-Dammer F, Kompa C, Bieser G, Jonsson T, Silva CM, Yang MM, Youvan DC, Michel-Beyerle ME (1996) Time-resolved spectroscopy of wild-type and mutant green fluorescent proteins reveals excited state deprotonation consistent with fluorophore-protein interactions. Chem Phys 213 1-16... 

Time-resolved spectroscopy establishes that the fluorescence of the excited (singlet) anthracene ( ANT ) is readily quenched by maleic anhydride (MA), which leads to the formation of the ion pair ANT+, MA via diffusional electron transfer (see Fig. 12), i.e.,... 

The role of the conditions in which these phenomena are observed is now well understood [40, 45], The chromophore should be solvatofluorochromic, that is, its fluorescence spectra should respond to changes in interaction energy with its environment by significant shifts. This environment should be relatively polar, but rigid or highly viscous, so that the relaxation times of its dipoles, tr, are comparable or longer than the fluorescence lifetime tf (in the case of recording the steady-state spectra) or on the time scale of observation (in time-resolved spectroscopy). Thus, these effects are coupled with molecular dynamics in condensed media. 

For the determination of the dissociation constant in the excited state, several methods have been used the Forster cycle,(109 m) the fluorescence titration curve/113 the triplet-triplet absorbance titration curve,014 but all involve the assumption that the acid-base equilibrium may be established during the lifetime of the excited state, which is by no means a common occurrence. A dynamic analysis using nanosecond or picosecond time-resolved spectroscopy is therefore often needed to obtain the correct pK a values.1(n5)... 

J.-C. Brochon, P. Wahl, M. Charlier, J. C. Maurizot, and C. Helene, Time-resolved spectroscopy of the tryptophanyl fluorescence, Biochem. Biophys. Res. Commun. 19, 1261-1271 (1977). 

Time-resolved luminescence spectroscopy may be extremely effective in minerals, many of which contain a large amount of emission centers simultaneously. With the steady state technique only the mostly intensive centers are detected, while the weaker ones remain unnoticed. Fluorescence in minerals is observed over time range of nanoseconds to milliseconds (Table 1.3) and this property was used in our research. Thus our main improvement is laser-induced time-resolved spectroscopy in the wide spectral range from 270 to 1,500 nm, which enables us to reveal new luminescence centers in minerals previously hidden by more intensive centers. 

The time-resolved spectroscopy is a sensitive tool to study the solute-solvent interactions. The technique has been used to characterize the solvating environment in the solvent. By measuring the time-dependent changes of the fluorescence signals in solvents, the solvation, rotation, photoisomerization, or excimer formation processes of a probe molecule can be examined. In conventional molecular solutions, many solute-solvent complexes. 

Time-Resolved Spectroscopy. Steady-state solvatochromic techniques provide a reasonable means to study solvation processes in supercritical media (5,17-32,43-45,59-68). But, unless the interaction rates between the solute species and the supercritical fluid are slow, these "static" methods cannot be used to study solvation kinetics. Investigation of the kinetics requires an approach that offers inherent temporal resolution. Fortunately, time-resolved fluorescence spectroscopy is ideally suited for this task. 

Stimulated fluorescence appears with a delay of about 500 fs relative to the A [ absorption, although both absorption and fluorescence stem from the same state, proved by decay measurements with picosecond time-resolved spectroscopy. This delayed appearance of stimulated fluorescence is caused by a quickly increasing and short-lived absorption A0 located in nearly the same spectral range as the fluorescence. The authors assumed that the instant absorption A0 is caused by a state located slightly below the level reached by excitation with 4eV. This state 2,... 

TNP-ATP complex obtained by the single-molecule time-resolved spectroscopy, together with a fluorescence decay curve of TNP-ATP obtained by a bulk measurement. Both curves were well fitted to biexponential functions. The instrument-response function in 195-ps fwhm is also displayed. (B) Representative fluorescence spectrums of two individual enzyme-TNP-ATP complexes showing different emission peaks. A fluorescence spectrum of TNP-ATP obtained from a bulk measurement is also displayed for comparison. All spectrums were normalized to unity at their maximum. (From Ref. 18.)... 

Experimentally, commercial steady-state fluorescence spectrometers can be equipped with polarizer attachments, either sheet or Glan-Thompson polarizers. Alternatively, sheet polarizers are usually easily incorporated into the sample cavity in the excitation and emission pathways. Likewise, for time-resolved spectroscopy, polarizers may simply be introduced into the excitation and detection paths. Frequently, the excitation source in time-resolved experiments is a laser which will be inherently polarized. 

It is well known that both nanometre and nanosecond-picosecond resolutions at an interface can be achieved by total internal reflection (TIR) fluorescence spectroscopy. Unlike steady-state fluorescence spectroscopy, fluorescence dynamics is highly sensitive to microscopic environments, so that time-resolved TIR fluorometry at water/oil interfaces is worth exploring to obtain a clearer picture of the interfacial phenomena [1]. One of the interesting targets to be studied is the characteristics of dynamic motions of a molecule adsorbed on a water/oil interface. Dynamic molecular motions at a liquid/liquid interface are considered to be influenced by subtle changes in the chemical/physical properties of the interface, particularly in a nanosecond-picosecond time regime. Therefore, time-resolved spectroscopy is expected to be useful to study the nature of a water/oil interface. 

Munro, I. H., Sabersky, A. P., Synchrotron Radiation as a Modulated Source for Fluorescence Lifetime Measurements and for Time Resolved Spectroscopy in Ref 10, p. 323... 

An important factor for in vivo studies is the auto fluorescent cellular background. To overcome this problem, pyrene-labeled binary probes have been used to take advantage of the long fluorescence lifetime (>40ns) of the pyrene excimer, compared with that of the cellular extracts (7 ns), that allows selective detection of the excimer using time-resolved spectroscopy (35). 

The time-resolved spectroscopy of Cr doped spinel and petalite-like phase types glass-ceramics enables a more accurate distinction of the and T2 energy levels of Cr(III) and of the equilibrium between the population of two levels, see Fig. 5, which presents the time-resolved spectra of spinel type glass-ceramics excited at 680 nm at room temperature Glass-ceramics derived from boro-silicate glasses containing Cr(III) were also recently studied by laser spectroscopy and fluorescence-line narrowing. 

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