Fluorescence spectra measurements

In contrast to the photosynthetic eukaryotes, photoprotection in cyanobacteria is not induced by the presence of a transthylakoid ApH or the excitation pressure on PSII. Instead, intense blue-green light (400-550 nm) induces a quenching of PSII fluorescence that is reversible in minutes even in the presence of translation inhibitors (El Bissati et al. 2000). Fluorescence spectra measurements and the study of the NPQ mechanism in phycobilisome- and PSII-mutants of the cyanobacterium Synechocystis PCC6803 indicate that this mechanism involves a specific decrease of the fluorescence emission of the phycobilisomes and a decrease of the energy transfer from the phycobilisomes to the RCs (Scott et al. 2006, Wilson et al. 2006). The site of the quenching appears to be the core of the phycobilisome (Scott et al. 2006, Wilson et al. 2006, Rakhimberdieva et al. 2007b). 

The fluorescence spectra measured just upon ablation are given in Figure 2A as a function of laser fluence. The contribution below 370 nm was suppressed, as a Hoya L37 filter was used in order to cut off the laser pulse. Fluorescence spectra of this polymer film consist of sandwich (max. 420 nm, lifetime 35 ns) and partial overlap (max. 370 nm, lifetime 16 ns) excimers (20). The latter excimer is produced from the initially excited monomer state, while the sandwich excimer from the partial overlap excimer and the monomer excited states. Since these processes compete with efficient interactions between identical and different excimers (Si - Si annihilation) (12), the sandwich excimer is quenched to a greater extent compared to the partial overlap one under a high excitation. Actually the fluence-dependent spectral change around the threshold can be interpreted in terms of Si - Si annihilation. 

Molecular fluorescence spectrometry has long been regarded as a useful technique for the determination of polycyclic aromatic hydrocarbons (PAHs) and related materials, due to the very high sensitivities which can be achieved. However, molecular fluorescence spectra measured in liquid solution usually are broad and relatively featureless hence, spectral interferences are common in the liquid-solution fluorometric analysis of multicomponent samples. Moreover, the fluorescence of a particular component of a complex sample may be partially quenched by other sample constituents if quenching occurs to a significant extent, the fluorescence signal observed for a particular compound present at a particular concentration will also depend upon the identities and concentrations of other substances present in the sample. Under these conditions, it is virtually impossible to obtain accurate quantitative results. Therefore, it is generally observed that molecular fluorescence spectrometry in liquid solution media is useful for quantitative determination of individual components in complex samples only if the fluorescence measurement is preceded by extensive separation steps (ideally to produce individual pure compounds or, at worst, simple two- or three-component mixtures). 

Fig. 18. (Left) Change in fluorescence spectra measured by streakscope at the different reaction times. The increasing count at 560 nm proved the formation of Zn(II)-ocqn complex at the interface of two-phase microsheath flow system. (Right) The result of the digital simulation of the Zn(II)-Hocqn complexation. The initial concentration of Hocqn was 1.1 x 10 4M.

Dependence of the observed excimer to monomer intensity ratio on polystyrene concentration for PS/PVME blends cast from tetrahydrofuran (circles) and toluene (squares) with the fluorescence spectra measured before (open symbols) and after (filled symbols) annealing at 383 K for 10 hours,... 

Conventional fluorescence spectra measured under the TIR condition and by the normal illumination are similar to fluorescence spectrum of N-ethylcarbazole in the B-film. This is because the latter was far thicker than the S-film. The time-resolved fluorescence spectra of 0.01 um S-film measured with an incident angle of 6, + 1.45° is shown in Fig. 2 (left). The... 

A large number of stilbenes derived from 3-(p-tolyl)indoxazene have been prepared and their fluorescence spectra measured.74... 

The kinetics of Pchlide photoreduction was previously reconstitued through 77 K fluorescence spectra measured after illumination at room temperature. Such kinetics were interpreted as a second order reaction (10) or as a sum of two first order reaction (11-12) These fluorescence measurements were influenced by the energy transfer from Pchlide to Chlide which has been shown to be very efficient at 77 K (13-14). Absorbance kinetics were also recorded (2,4,10) but on a very extended time scale (up to Ih) during which spectral shifts of Chlide and Pchlide regeneration occur. 

Fig. 17 shows three examples of fluorescence spectra measured at different distances from the surface. At 1.2 pm only statistical fine structure can be observed. In this case the illuminated area is very large and many molecules with the same resonance frequency are excited. Their fluorescence overlaps and it is not possible to distinguish individual molecules. Nevertheless, the fine structure spectrum is reproducible, as demonstrated by the two traces taken 5 min. apart. Closer to the surface the single molecule features become more pronounced. The excited volume is now smaller and... 

Figure 3.15 ESDPT in dipyridocar-bazole alcohol complexes, (a) Structure of dipyrido[2,3-a 3, 2 -i]carbazole (32) and its fluorescence spectra measured in n-propyl alcohol at 298 K (top) and 123 K (bottom) [69]. (b) Fluorescence transients of 32 (in 1-propanol) detected at the wavelengths indicated. Transients for F, emission band (left) and (right) transients for Fj emission...

Figure 14. (Upper panel) The ruby fluorescence spectrum measured in quasi-hydrostatic conditions at 7.7 GPa. (Lower panel) The empirical law describing ruby Ri line shift with pressure [96] is also reported.

Figure 17. Time-resolved fluorescence spectra of a solute with one vibrational mode in ethanol at 247 K.68 The various frames show the fluorescence spectrum measured at successively later times after the application of a 1 ps excitation pulse. Each spectrum is labeled with the observation time. The steady-state fluorescence spectrum is given by the dashed curve in the bottom frame. In the electronic ground state, the solute vibrational frequency is400cm 1, and in the excited state, the frequency is 380 cm 1. The dimensionless displacement is 1.4. The permanent dipole moment changes by 10 Debye upon electronic excitation. The Onsager radius is 3A. The longitudinal dielectric relaxation time, xL, is 150 ps.

Antibiotics were used in folk medicine at least as early as 2500 years ago when the Chinese reported the medicinally beneficial effects of moldy bean curd Evidence for some type of tetracycline antibiotic usage by the Sudanese-Nubian civilization (350 AD) was reported in 1980 (6). Fluorescent areas in human bones from this era were observed that were identical in location and characteristics to modem bone from patients treated with tetracyclines. Identification of tetracycline in the ancient bones was further substantiated by fluorescence spectrum measurements and microbiological inhibition studies (7). 

An important extension to the simplest upconversion experiment at a single detection frequency M2 is the practice of measuring time-resolvedfluorescence spectra, that is, the shape of the fluorescence spectrum... 

Tabic 6-5. Comparison of (he aK vibrational modes in the ground and excited states. The totally symmetric vibrations of the ground stale measured in tire Raman spectrum excited in pre-resonance conditions 3S] and in the fluorescence spectrum ]62 ate compared with the results of ab initio calculations [131- The corresponding vibrations in the excited stale arc measured in die absorption spectrum. 

Fig. 6.1.5 Fluorescence spectra of the purple protein (1-4) and the luminescence spectrum measured with Latia luciferin, luciferase and the purple protein (5 Xmax 536 nm). Excitation spectra (1) and (2) were measured with emission at 630 nm and 565 nm, respectively. Emission spectra (3) and (4) were measured with excitation at 285 nm and 380 nm, respectively. From Shimomura and Johnson, 1968c, with permission from the American Chemical Society.

Fig. 6.2.5 Fluorescence spectra of pholasin after treatment with 5M guanidine hydrochloride. Left, excitation spectrum measured at 460 nm right, emission spectrum measured with excitation at 360 nm. From Henry et al., 1973, with permission from Elsevier.

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

Radiation from a xenon or deuterium source is focussed on the flow cell. An interchangeable filter allows different excitation wavelengths to be used. The fluorescent radiation is emitted by the sample in all directions, but is usually measured at 90° to the incident beam. In some types, to increase sensitivity, the fluorescent radiation is reflected and focussed by a parabolic mirror. The second filter isolates a suitable wavelength from the fluorescence spectrum and prevents any scattered light from the source from reaching the photomultiplier detector. The 90° optics allow monitoring of the incident beam as well, so that dual uv absorption and fluorescence... 

Fig. 6 Normalized linear absorption (solid black line), anisotropy (blue circles), and corresponding 2PA spectrum measured by two-photon fluorescence (red squares) and Z-scan (green circles) for SD 2405. Molecular structure is shown to the left...

Exciplex methodhas also been proposed for droplet temperature measurement. In an oxygen environment, however, the fluorescence from the exciplex is quenched by the oxygen. In addition, fuel droplets may contain aromatic hydrocarbons that can produce fluorescence emissions, masking the fluorescence spectrum of the dopants used for the temperature determination. 

The Na+ sensor M-9 has a structure analogous to that of compound E-4, but instead of two identical pyrene fluorophores, it contains two different fluorophores with a pyrene group and an anthroyloxy group. Resonance energy transfer (see Chapter 9) from the former to the latter is then possible because of the spectral overlap between the fluorescence spectrum of the pyrene moiety and the absorption spectrum of the anthroyloxy moiety. Upon addition of Na+ to a solution of M-9 in a mixture of MeOH and THF (15 1 v/v), the fluorescence of the anthroyloxy group increases significantly compared with that of the pyrene group, which permits a ratiometric measurement. 

The fluorescence spectrum of the tris-acridine cryptand A-13 shows the characteristic monomer and excimer bands. Upon complexation with various organic anions (carboxylates, sulfonates, phosphates), the monomer band increases at the expense of the excimer band. The stability of the complexes depends on the contribution of the electrostatic and hydrophobic forces and on the structural complementarity. Stability constants of the complexes ranging from 103 to 107 have been measured. In particular, A-13 binds tightly to mono- and oligonucleotides, and it can discriminate by its optical response between a pyridimic and a purinic sequence. 

For the distyrylbenzene carbon-centered tetramer 46b, the fluorescence spectrum in the solid him differs from the spectra in solution or in a polymer matrix due to excimer formation [93]. A concentration of 5% in a polystyrene matrix is sufficient for a distinct broadening of the emission. For the higher homologue 46c, a fluorescence maximum of 472 nm was measured in freshly prepared films. If the film is thermally annealed, the spectrum shifts to 511 nm, probably due to intermolecular arrangement that favors excimer formation. 

Figure 8.5. Palm of the hand. Fluorescence spectrum (Fb-mode, full line) and diffuse reflectance absorption spectrum (dashed line, normalized to X = 700 nm, reference filter paper) measured with a sensitized diode array spectrometer.

Measurement of the decay kinetics /(/) in different regions of the fluorescence spectrum. If relaxation (or any reaction in the excited state) is absent, I(t) does not depend on vem, whereas in its presence, the spectral dependence illustrated in Figure 2.7 is observed. 

Native fluorescence of a protein is due largely to the presence of the aromatic amino acids tryptophan and tyrosine. Tryptophan has an excitation maximum at 280 nm and emits at 340 to 350 nm. The amino acid composition of the target protein is one factor that determines if the direct measurement of a protein s native fluorescence is feasible. Another consideration is the protein s conformation, which directly affects its fluorescence spectrum. As the protein changes conformation, the emission maximum shifts to another wavelength. Thus, native fluorescence may be used to monitor protein unfolding or interactions. The conformation-dependent nature of native fluorescence results in measurements specific for the protein in a buffer system or pH. Consequently, protein denatur-ation may be used to generate more reproducible fluorescence measurements. 

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