Luminescence Probing Methods

Luminescence probes are molecules or ions which, upon photoexcitation, emit light having characteristics sensitive to the immediate environment of the probe [243]. The characteristics of emitted light serve to characterize the environment of the luminescence probe. 

Luminescent probes can be divided into two groups fluorescence probes and phosphorescence probes. Fluorescence is an emission of light associated with 

Some molecules or ions can function as quenchers and inhibit luminescence. Quenching, caused by interactions between the luminescent probe and a quencher, may be reversible or irreversible. Excimer formation is a case of reversible quenching. Some luminescent probes react, in the excited state, with an identical molecule in the ground state and form an excimer  

Another reversible quenching technique used in micellar studies is energy transfer from the probe in the excited state to an energy-acceptor molecule. 

The luminescence methods involve solubilization of the probe in micelles and the determination of fluorescent spectra and fluorescence polarization. Various steady-state and transient-state fluorescence methods have been employed. Experimental details can be found in the literature [246-250]. A careful selection of the probe is essential for obtaining meaningful results. Luminescence methods are based on the assumption that the probe does not affect the fundamental nature of the solution and the micelles. This assumption must be validated for the system being studied. 

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Valeur B. (1993) Fluorescent Probes for Evaluation of Local Physical, Structural Parameters, in Schulman S. G. (Ed.), Molecular Luminescence Spectroscopy. Methods and Applications, Part 3, Wiley-Interscience, New York, pp. 25-84. 

B. Valeur, Fluorescent probes for evaluation of local physical and structural parameters, in Molecular Luminescence Spectroscopy, Methods and Applications Part 3 (S. G. Schulman, ed.), pp. 25-84, John Wiley Sons, New York (1993). 

Electronic Absorption and Luminescence (Volume 12) Absorption and Luminescence Probes Fluorescence Imaging Microscopy Fluorescence Lifetime Measurements, Applications of Indirect Detection Methods in Capillary Electrophoresis Surface Measurements using Absorption/Luminescence... 

Eu(Tc)(cit) is 1 1 2 in this case. The fluorescence intensity of the 615-nm emission line of [Eu(Tc)(cit)2 ] is 22 times stronger than that of [Eu(Tc)]. Citrate, as a polydentate ligand, can chelate the Eu3+ ion via the oxygen atoms of its carboxy and hydroxy groups. It is assumed that citrate displaces water molecules which ligate to the 8- and/or 9-coordination sites of the Eu3+ ion and quench its fluorescence. Table 5 summarizes the luminescence properties of the [Eu(Tc)] complex (1 1 stoichiometry) and the corresponding chelate complexes with the intermediates of the citrate cycle. Citrate concentrations can be imaged with this luminescent probe by means of the RLI method (Fig. 15) [107]. 

A and B are constants that can be determined for a given luminescent probe (i.e., Eu or Tb). Various values of A and B can be found in the literature and are presented in table 4. In this table, each expression is only referenced once, although it may have been used in numerous papers. More sophisticated expressions (see sect. 3.6.4) have not been included. Table 4 also displays the numerical values used in eq. (12b) (derived from Horrocks and Sudnick (1983)) for Eu(III) and Tb(III). The studies by Bryden and Reilley (1982), Wang and Horrocks (1997), Wang et al. (1999), already cited in the experimental section and others (Albin et al., 1984 Barthelemy and Choppin, 1989 Shin and Choppin, 1999), correspond to this approach, either in its exact (eq. (12b)) or simplified (eq. (13)) version. Finally, note that the authors using this method ascribe the decay rate changes mostly to the water molecules but that some authors use the more general term of OH bonds . 

TRES is a valuable method to determine rate constants in the very specific case of a reaction between an excited species and a quencher. The theoretical approaches are easily applicable and lead to interesting results on various aspects of such reactions. Another type of reaction rate constant can be studied by TRES but is rather anecdotal in the case where a ligand, L, reacts very slowly (on the order of hours to months) with the luminescent probe, the formation of the complex can be followed by TRES (for example, see Wu et al. (1996), Bazzicalupi et al. (2001)). 

The Stern-Volmer equation (see sect. 4) may be used to determine small amounts of a species which would behave as an inhibitor of a given luminescent probe. The detection limit depends, among other parameters, on and on the detection limits of the setup. The potentials of this method for analytical purposes are discussed, on a general aspect, in Borissevitch (1999), Rakicioglu et al. (1998) and the specific cases of Eu(III) or U(VI) are presented in Georges (1993), Lopez and Birch (1996), Kessler (1998). For example, a detection limit of 7 ngl-1 for Cu2+ is obtained (Lopez and Birch, 1996). Numerous factors may render the method difficult to apply besides the variations of ksv as a function of ionic strength, if more than one quencher is present in solution, it becomes difficult to determine their individual concentrations. This problem has been studied in the case of solutions that more or less mimic the nuclear fuel solutions in Katsumura et al. (1989). 

Hanaoka, K., Kikuchi, K., Kobayashi, S., and Nagano, T. (2007) Time-resolved long-lived luminescence imaging method employing luminescent lanthanide probes with a new microscopy system. Journal of the American Chemical Society, 129, 13502-13509. 

All three methods give similar values of interfacial potentials typical results for some of micelles and vesicles are listed in Table 3. Also listed are estimates of interfacial dielectric constants (e), determined by comparing the position of absorption bands of solvatochromic indicators in the surfactant assemblies with that of reference 1,4-dioxane water mixtures with known e values. More generally, luminescence probe analysis [49], thermal leasing [50] and absorption spectroscopy [47, 51] are techniques that have all been utilized to measure local polarities in micelles and vesicles. It is important to note that these methods presume knowledge of the loca-... 

Applications of photophysics in biology and medicine are very extensive and only a few topics can be mentioned in this review. A survey of the use of lanthanide ions as luminescent probes of biomolecular structure and a general account of long distance electron transfer in proteins and model systems are very helpful. The methods applicable to the synthesis and activation of a number of photoactivable fluoroprobes have been described and photoactivation yields measured . 

The application of luminescence techniques to the study of macro-molecular behaviour has enjoyed an enormous growth rate in the last decade. The attraction of such methods lies in the degrees of both specificity and selectivity afforded to the investigator. Consequently the polymer may be doped or labelled at sufficiently low concentration levels of luminophore as to induce minimal perturbation of the system. Polarized photoselection techniques offer particular attraction in the study of relaxation phenomena both in solution and solid states. In principle, astute labelling can allow elucidation of the molecular mechanisms responsible for the macroscopic relaxations exhibited by the raacromolecular system. In addition, luminescent probes can address the microviscosity of their environment. 

A method based on fluorescence quenching that did not depend on the nature of the transition was used to determine the micelle size of the hexyl copolymer (24). The basic idea underlying this method is that, in a solution containing luminescent probe and quencher molecules, both solubilized in an excess of micelles, the quenching will be inversely related to the number of micelles, because the more micelles there are, the smaller is the chance of both a probe and a quencher molecule inhabiting the same micelle (25-27). The hexyl copolymer used in our study had a degree of polymerization of 1700. The fluorescent probe was tris(2,2 -bipyridine)ruthenium(II) ion [Ru(bpy)3 ], the quencher was 9-methylanthracene (9-MeA), and the solvent was an aqueous 0.1 M LiCl solution. The fluorescence experiments were supplemented with solubilization experiments from these, the distribution of the 9-MeA between the polymer molecules and the solvent molecules, as well as the extent to which the polymer was in micellar form, could be simultaneously determined. The results indicated that the micelles inside the domain of a macromolecule encompassed approximately 24 repeat units, and that this micelle size was independent of the polymer concentration, of the probe concentration, and the extent to which the polymer was micellized. 

The FRET process between donor and acceptor luminescent probes is a versatile spectroscopic tool to follow the intimate contact between molecular components. Hence, it also serves as a useful method to follow sensing events between recognition elements and their analytes [53-55]. 

The most important applications of luminescence probing in microemulsions involve the deactivation dynamics or excitation energy transfer properties of the excited states. With a brief flash of light a population of excited species is created in the sample, and the subsequent deactivation is observed over time. The decay of the excited probe, and the fluorescence spectrum, may depend on the interactions with the environment, which reveal useful information. In time-resolved luminescence quenching (TRLQ), however, it is the interaction of the probe with another added component, a quencher, that is studied. This method is dealt with here. For micellar systems, several publications have already discussed it in both experimental and theoretical detail [1-6]. 

In this chapter, we concentrate on luminescence approaches that are most suitable for stilbenes to be molecular probes and are based on specific and nonspecific labeling, competition, solvatochromism, experimental molecular dynamics, and singlet-singlet and triplet-triplet energy transfer. A general survey is made of the physical principles and application of the fluorescence probe methods stressing on latest developments in this area. 

The luminescent lifetime of the anhydrous compound has also been studied but no definitive result was obtained. The authors concluded this paper saying that the anhydrous microporous phase presenting ID channels can be potentially considered as luminescent probe for detecting small molecules. That is the reason why two years later another group published another paper on this family. In this paper (Pan et al., 2001) the synthesis, via hydrothermal method, and the crystal stracture (see fig. 12) of a compound with chemical formula Er4(bdc)6-6H20 are described. 

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