Time-resolved fluorescence spectrum

Evolution of fluorescence spectra during the lifetime of the excited state can provide interesting information. Such an evolution occurs when a fluorescent compound is excited and then evolves towards a new configuration whose fluorescent decay is different. A typical example is the solvent relaxation around an excited-state compound whose dipole moment is higher in the excited state than in the ground state (see Chapter 7) the relaxation results in a gradual red-shift of the fluorescence spectrum, and information on the polarity of the microenvironment around a fluorophore is thus obtained (e.g. in biological macromolecules). 

In phase fluorometry, the phase (and modulation) data are recorded at a given wavelength and analyzed in terms of a multi-exponential decay (without a priori assumption of the shape of the decay). The fitting parameters are then used to calculate the fluorescence intensities at various times, 2 3 The procedure is repeated for each observation wavelength X, X2, A3. It is then easy to reconstruct the spectra at various times. 

In pulse fluorometry, we take advantage of the fact that the amplitudes of the output pulses of the TAC are proportional to the times of arrival of the fluorescence photons on the photomultiplier. Selection of a given height of pulse, i.e. of a given time of arrival, is electronically possible (by means of a single-channel analyzer) and allows us to record the fluorescence spectra at a given time t after the excitation pulse. This is repeated for various times. The method described above for phase fluorometry can also be used in pulse fluorometry. 

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Murakami H, Kinoshita S, Hirata Y, Okada T and Mataga N 1992 Transient hole-burning and time-resolved fluorescence spectra of dye molecules in solution evidence for ground-state relaxation and hole-filling effect J. Chem. Phys. 97 7881-8... 

Another powerful tool for examining this issue is the use of time-resolved fluorescence spectra, especially when combined with the technique of Time-Resolved Area Normalized Emission Spectra (TRANES) developed by Periasamy and coworkers [78-80]. In this method, separate decay curves are collected over a wide range of emission wavelengths and reconstructed into time-resolved spectra, which are then normalized to constant area. In this model-free approach, it is possible to deduce the nature of heterogeneity of the fluorescent species from the... 

WAVELENGTH (nm) Figure 19. Time-resolved fluorescence spectra for J-aggregate LB film of [CI-MC] [AA] = 1 2. 

The principle of the determination of time-resolved fluorescence spectra is described in Section 6.2.8. For solvent relaxation in the nanosecond time range, the single-photon timing technique can be used. The first investigation using this technique was reported by Ware and coworkers (1971). Figure 7.3 shows the reconstructed spectra of 4-aminophthalimide (4-AP) at various times after excitation. 

Figure 8.9. Time-resolved fluorescence spectra of 9,10-diphenylanthracene, recorded wi th time-correlated single photon counting, Aa = 360 nm. Parameters gate width and delay time relative to the intensity maximum of the excitation pulse.

Figure 12.22 shows the time-resolved fluorescence spectra of a synthetic bacteria (.R. Sphaeroides) up to 1000 nm at gate widths of 5 psec, recorded using an MCP-PM with an SI photocathode.(88)... 

I. Yamazaki, M. Mimuro, N. Tamai, T. Yamazaki and Y. Fujita, Picosecond time-resolved fluorescence spectra of photosystem I and II in Chlorella pyrenoidosa, FEES Lett. 179, 65-68 (1985) and references therein. 

L. Brand and J. R. Gohlike, Nanosecond time-resolved fluorescence spectra of a protein-dye complex, J. Biol. Chem. 246, 2317-2324 (1971). 

Figure 41. Time-resolved fluorescent spectra in the piperidine adduct of a four-ligand europium dibenzoylmethide chelate [from Ref. (63)].

Fig. 2. Time resolved fluorescence spectra of all-trans PRSB in methanol (black) and octanol (grey) for a) t<50 fs and b) t>50 fs. The intensity of the octanol spectra is adjusted the methanol spectra. The spectra are not corrected for self-absorption (for >19.500 cm 1), or for the detector response function. A residual signal appearing at energies <14.000 cm"1 is due to incomplete background subtraction (see above).

Fig. 2 (a) Fluorescence up-conversion transients of TAB dissolved in n-heptane. Detection wavelengths in nm are indicated in circles (b) reconstructed time-resolved fluorescence spectra of TAB in n-heptane... 

Figure 29. Time-resolved fluorescence spectra of BA in acetone at ambient temperature. The points in the figure are experimental data. The curves through the data are log normal function [106] fits to the data. 

Figure 30. Simulated time resolved fluorescence spectra for BA in propylene carbonate. See text for further details.

The above described model sequences have been studied both as oligomers [7,8,11-13,19] and as polymers [9,11,20]. An increase in the size of the helix is known to reinforce its stability, as revealed by their melting curves [18] and attested by X-ray diffraction measurements in solution [21]. Therefore, in this chapter we focus on the polymeric duplexes poly(dGdC).poly(dGdC) [= 1000 base-pairs], poly(dAdT).poly(dAdT) [= 200-400 base-pairs] and poly(dA).poly(dT) [= 2000 base-pairs] studied by us. First we discuss the absorption spectra, which reflect the properties of Franck-Condon states, in connection with theoretical studies. Then we turn to fluorescence properties fluorescence intensity decays (hereafter called simply fluorescence decays ), fluorescence anisotropy decays and time-resolved fluorescence spectra. We... 

Fig. 6 Time-resolved fluorescence spectra obtained for poly(dGdC).poly(dGdC) (green), poly(dAdT).poly(dAdT) (blue) and poly(dA).poly(dT) (red) in phosphate buffer at zero-time. Circles correspond to experimental data and solid lines to fits with log-normal functions. Excitation wavelength 267 nm.

The ensemble of the experimental results briefly reviewed here, e.g. steady-state absorption and fluorescence spectra, fluorescence decays, fluorescence anisotropy decays and time-resolved fluorescence spectra, allow us to draw a qualitative picture regarding the excited state relaxation in the examined polymeric duplexes. Our interpretation is guided by the theoretical calculation of the Franck-Condon excited states of shorter oligomers with the same base sequence. 

Figure 1.4. Time resolved fluorescent spectra of a dual dansyl-nitroxide probe 1 incorporated in bovine serum albumin (Likhtenshtein et al., 2000a). Reproduced with permission.

The CC2 calculation indicates that the emission maximum, corresponding to the vertical transition from the tttt minimum to the electronic ground state, lies at longer wavelength for conformer B (—440 nm) than for conformer A ( 400 nm) [24], This would lead to a dual fluorescence with the picosecond (30 ps) component having longer-wavelength emission relative to the nanosecond (12 ns) component. Consistent with this prediction, the time-resolved fluorescence spectra of PdG, taken at short times (0-100 ps) has emission peak at about 440 nm, whereas that taken at... 

This controlled dye array can be mimicked using thin film preparation techniques. The LB method is the technique most suited to layering various dye molecules in a desired sequence and at desired distances. Figure 6.8 shows hetero-type LB films that can mimic the dye array in the photosynthetic system of a cyanobacterium. Detailed time-resolved fluorescence spectra upon... 

It is well known that the fluctuational behavior of the solvent molecules is characterized by the normalized time correlation function of the fluctuation, p (r), which is expressed as a following equation using observed time resolved fluorescence spectra. 

Figure 2. Reconstructed time-resolved fluorescence spectra of Cl02 in (a) DMA and (b) AN. TTte dots are the dam experimentally obtained, and the curves are the log-normal fits to the dots.

Time-resolved fluorescence spectra can be obtained without recourse to sophisticated equipment by the use of an electronic quencher, (invariably molecular oxygen), wdiich will quench preferentidly those mdecules with the longest decay times, as is illustrated by the Stern-Volmer Eq. (61). Addition of increasing amounts of quencher to a system under continuous illumination thus progressively... 

It is of interest to note that within the limit of acoiracy of these experiments, monomer decay curves (Fig. 22) were single exponential, whereas Sdieme 1 predicts dual exponentiality (Eq. 65). The results thus imply that in pdyslyrene reverse dissociation ( feedback ) of the excimer is not of importance. This point is amplified by time-resolved fluorescence spectra which show that late- ted >ecti a (see experimental section) are composed exclusively of excimer emission (Fig. 23). The same is true in poly(a-methylstyrene) In view of more recent work mi other vinyl aromatic ptdymers, it would be of interest to study pdy(styrene) further with more sophisticated techniques. 

Fig. 23. Gated time-resolved fluorescence spectra of atactic poly (styrene) in dichloromethane solution, (a) Early gated spectrum, delay St = 0 ns, gate width St = 3 ns. (b) Late gated spectrum, St =45 ns, St = 3 ns...

It will be noted that the kinetic scheme adapted above reverse dissociation of excimer sites, the experimental evidence for this being the triple exponential decay of the monomer fluorescence and the gated time-resolved fluorescence spectra, reported here and seen earlier " in the homopolymer. 

The system 2-naphthylamine/triethylamine forms proton donor/acceptor interaction, which was investigated in the excited state by measuring time-resolved fluorescence spectra. While the similar 2-naphthol/triethylamine system affords the ion pair interaction, via the hydrogen bond complex, the 2-naphthylamine/triethylamine system presents hydrogen bond interaction which shows a low-temperature absorption with 7max = 370 nm, and Amax = 370 nm in the fluorescence spectrum148. 

The transition dipole moment of BChl c-744 is nearly parallel to the axis of the rod element, while that of BChl c-727 is more random. The presence of the longer-wave length BChl c-complex, BChl c-766, has been suggested by the results of deconvolution of the linear-dichroism spectra as well as by more recent measurements of time-resolved fluorescence spectra of oriented chlorosomes. The orientation of the transition moment of BChl c-766, determined from its fluorescence maximum at 778 mn, is intermediate between that of BChl c-744 and that of the baseplate BChl c-protein complex, B795. 

Fig. 6. Time-resolved fluorescence spectra of Cf. aurantiacus excited at 715 nm. Numbers on right margin indicate time of observation relative to the arrival time of the peak of the excitation pulse in ps. Dashed-line spectrum in the bottom frame shows the absorption spectrum of intact cells. Figure source Mimuro (1990) Studies on excitation energy flow in the photosynthetic pigment system structure and energy transfer mechanism. Bot Mag, Tokyo. 103 248.

Time-resolved fluorescence spectra clearly indicate a linear cascade of excitation-energy transfer from BChl c in the chlorosome rods, through the BChl a-containing baseplate, to the core-antenna BChl a in the membrane, and finally to the reaction center. Overall excitation transfer efficiency from BChl c to the core antenna (B806-866) in Cf. aurantiacus cells has been reported to be 69 13% at 50 °C, but only -15% at 4 K. Excitation transfer from B806 to B866 within the core antenna is 100%. When the reaction center is closed, the 883-nm fluorescence decays less rapidly in -250 ps. 

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