Time resolved fluorescence methods

Kolb, A.J., Burke, J.W., and Mathis, G., Homogeneous, time-resolved fluorescence method for drug discovery, in High Throughput Screening, The Discovery of Bioactive Substances, Devlin, J.P., Ed., Marcel Dekker, New York, 1997, chap. 19. 

It is clear that in all cases mentioned above, time-resolved fluorescence methods have enabled a far greater insist into the character and complexities of biopolymer luminescence than have previously been obtained by steady state illuminatnn techniques. 

However, it was the microviscosity, rather than the bulk viscosity which was probed by using picosecond time-resolved fluorescence methods, since the value of C was constant within a homologous series of solvents but was different for monofunctional and difunctional alcohols. The probe molecule has a natural (radiative) lifetime, Xp of 4.6ns, so the reduction in lifetime results from the competition between fluorescence ( p 2xl0 s ) and the high rate of internal conversion (A ic lO" s ). This was attributed to rotation of the N-tolyl group of the dye, which then has to sweep through the solvent and hence probes the local environment. 

Fluoroimmunoassay makes use of the above behaviour. One of the common commercial methods is dissociation-enhanced fluoroimmunoassay (DELFIA). In this, a nonfluorescent Eu(III) EDTA-like complex is attached by a simple chemical reaction to an antibody or antigen, in a process called labelling. An immunoreaction is next initiated to bind the target, and then a (3-diketone and trioctylphosphine oxide (TOPO) mixture are added to the immunocomplex formed, at pH 3, to promote release of the Eu(III) from the antibody and its complexation as the strongly fluorescent complex [Eu((3-diketonate)3(TOPO)2], which is then measured by time-resolved fluorescence methods. The signal size relates to the amount of europium complexed, which in turns relates directly to the amount of the specifically formed target immunocomplex. This process is represented schematically in Figure 9.5. 

Time-resolved fluorescence methods are discussed elsewhere in this volume, and we shall not enter into lengthy descriptions of the technique but rather will point out some interesting aspects of time-resolved anisotropy methods as they apply to binding studies. The two principal time-resolved methodologies presently utilized are the impulse-response... 

Kolb, A., Burke, J., Mathis, G. (1997). Homogeneous, Time-Resolved Fluorescent Method for Drug Discovery. In High Throughput Screening The Search for Bioactive Molecules. J. Devlin (ed) pp 345-360. Marcel Dekker, New York. 

Time-resolved fluorescence is perhaps the most direct experunent in the ultrafast spectroscopist s palette. Because only one laser pulse interacts with the sample, the mediod is essentially free of the problems with field-matter time orderings that arise in all of the subsequently discussed multipulse methods. The signal... 

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... 

It is confirmed that the polymer matrix around ablated area was also affected strongly by laser ablation. The change of the matrix properties are brought about over a few tens of pin. This type of information is basically important and indispensable for practical applications such as excimer laser lithography. The time-resolved fluorescence spectroscopy is one of the powerful characterization methods for ablated polymer matrix. 

Recent developments in laser technology and fast detection methods now allow the kinetic behaviour of the excited state species arising from absorption of radiation by polymers to be studied on time-scales down to the picosecond region ( ). An example of a time-resolved fluorescence spectrometer which can be used to study such ultrafast phenomena is illustrated in Figure 5 Q). 

The choice of method depends on the system to be investigated. The methods of intermolecular quenching and intermolecular excimer formation are not recommended for probing fluidity of microheterogeneous media because of possible perturbation of the translational diffusion process. The methods of intramolecular excimer formation and molecular rotors are convenient and rapid, but the time-resolved fluorescence polarization technique provides much more detailed information, including the order of an anisotropic medium. 

E. P. Diamandis and R. C. Morton, Time-resolved fluorescence using a europium chelate of 4,7-bis(chlorosulfophenyl)-l,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Labelling procedures and applications in immunoassays, J. Immunol. Methods 112, 43-52 (1988). 

The dipole-dipole interactions of the fluorophore in the electronic excited state with the surrounding groups of atoms in the protein molecule or with solvent molecules give rise to considerable shifts of the fluorescence spectra during the relaxation process. These spectral shifts may be observed directly by time-resolved spectroscopic methods. They may be also studied by steady-state spectroscopic methods, but in this case additional data must be obtained by varying factors that affect the ratio between tf and xp. 

The broad field of nucleic acid structure and dynamics has undergone remarkable development during the past decade. Especially in regard to dynamics, modem fluorescence methods have yielded some of the most important advances. This chapter concerns primarily the application of time-resolved fluorescence techniques to study the dynamics of nucleic acid/dye complexes, and the inferences regarding rotational mobilities, deformation potentials, and alternate structures of nucleic acids that follow from such experiments. Emphasis is mainly on the use of time-resolved fluorescence polarization anisotropy (FPA), although results obtained using other techniques are also noted. This chapter is devoted mainly to free DNAs and tRNAs, but DNAs in nucleosomes, chromatin, viruses, and sperm are also briefly discussed. 

Figure 4.1. Time scales for rotational motions of long DNAs that contribute to the relaxation of the optical anisotropy r(t). Experimental methods used to study these motions in different time ranges are also indicated along with the authors and dates of some early work in each case. FPA, Fluorescence polarization anisotropy (Refs. 15, 18-20, and 87) TPD, transient photodichroism (Refs. 28 and 62) TEB, transient electric birefringence (Refs. 26 and 27) DDLS, depolarized dynamic light scattering (Ref. 116) TED, transient electric dichroism (Refs. 25, 115, and 130) Microscopy, time-resolved fluorescent microscopy (Ref. 176).

M. G. Badea and L. Brand, Time-resolved fluorescence measurements, Methods Enzymol. 61, 378-425 (1979). 

In the past ten years, numerous applications of fluorescence methods for monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted the development of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation for lifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician s office testing, future innovations in fluorescence immunoassay development will be expected to center on the simplification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing. 

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