Time-resolved fluorescence analyses

Perozzo, M. A., Ward, K. B., Thompson, R. B., and Ward, W. W. (1988). X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals. J. Biol. Chem. 263 7713-7716. 

Fluorescence polarisation studies on acenaphthylene labelled polyacrylic acid has demonstrated complexation occurs with polyethylene oxide to give a tightly packed and sterically restricted conformation SS. Neutralisation of the acidic groups restored the polarisation of the fluorescence and together with time resolved fluorescence analysis indicated that the motion of the probe is anisotropic. Solvent partitioning has been observed between glycol methacrylate copolymers from the fluorescence anisotropy changes of... 

The steady-state anisotropy can be related to the molecular motion by time-resolved fluorescence analysis, both reported in Fig. 4 and in Table 2. 

Cross A J and Fleming G R 1984 Analysis of time-resolved fluorescence anisotropy decays Blophys. J. 46 45-56... 

E. Analysis of Interfacial Complex by a Time-Resolved Fluorescence Spectroscopy... 

Samiotaki M, Kwiatkowski M, Ylitalo N, Landegren U (1997) Seven-color time-resolved fluorescence hybridization analysis of human papilloma virus types. Anal Biochem 253 156-161... 

Lakowicz J. R. and Szymacinski H. (1996) Imaging Applications of Time-Resolved Fluorescence Spectroscopy, in Wang X. F. and Herman B. (Eds), Fluorescence Imaging Spectroscopy and Microscopy, Chemical Analysis Series, Vol. 137, John Wiley ... 

Thus, the rs is a complex term which embodies the fluorescence lifetime, rotational correlation time (0), and also r0 (r1 in the absence of depolarizing motion). The most common type of experiment involves a comparison of rs for two experimental conditions however, such a comparison of rs ignores possible changes in x, , and r0. Nevertheless, for many cases a comparison of rf values alone may be satisfactory although a more rigorous analysis requires a time-resolved measurement. A comparison of the effects of changes in common membrane properties on time-resolved fluorescence parameters is shown in Table 5.3. 

Extensions of the analysis of time-resolved fluorescence anisotropy decay data in terms of two order parameters have also been developed (see, e.g., Refs. 51-54). Thus, the corresponding higher order parameter term is <7%) given by(53)... 

For PSI core it has been argued that excitation visits P700 2-3 times on an average prior to being trapped [221]. This conclusion is based on the analysis of time resolved fluorescence trapping times as a linear function of antenna size in terms of the Pearlstein model array treatment (see Sect. 5). A similar conclusion... 

Klostermeier, D. and D. P. Millar. Time-resolved fluorescence resonance energy transfer A versatile tool for the analysis of nucleic acids. Biopolymers 61, 159-179 (2002). 

In this paper, we present a preliminary analysis of the steady-state and time-resolved fluorescence of pyrene in supercritical C02. In addition, we employ steady-state absorbance spectroscopy to determine pyrene solubility and determine the ground-state interactions. Similarly, the steady-state excitation and emission spectra gives us qualitative insights into the excimer formation process. Finally, time-resolved fluorescence experiments yield the entire ensemble of rate coefficients associated with the observed pyrene emission (Figure 1). From these rates we can then determine if the excimer formation process is diffusion controlled in supercritical C02. 

Figure 8 shows a pair of typical time-resolved fluorescence decay traces for 100 / M pyrene in supercritical CO2 (Tr = 1.02 pr = 1.17). Note that the ordinate is logarithmic. The upper and lower panels show results for selective observation in the monomer (400 +. 10 nm) and excimer (460 + 10 nm) regions of the pyrene emission spectrum. Several interesting features are apparent from these traces. First, both decay processes are not single exponential. Second, the excimer emission has a significant contribution from a species that "grows in" between 30 - 75 ns this is a result of the excimer taking time to form (i.e., k in Figure 1). Third, the fits between the experimental data and the model shown in Figure 1 are good. Detailed analysis of these decay traces (10,11,21-26) yields the entire ensemble of photophysical kinetic parameters for the pyrene excimer in supercritical C02.

Reimer, G.J., S.J. Gee, and B.D. Hammock (1998). Comparison of a time-resolved fluorescence immunoassay and an enzyme-linked immunosorbent assay for the analysis of atrazine in water. J. Agric. Food Chem., 46 3353-3358. 

Figure 8.9 Time-resolved fluorescent lifetime analysis of Cy3 attached to double-stranded DNA (Iqbal et al., 2008b). Fluorescent decay curve for Cy3 attached to a 16 bp DNA duplex, showing the experimental data and the instrument response function (IRF), and the fit to three exponential functions (line). The decay curve was generated using time-correlated single-photon counting, after excitation by 200 fs pulses from a titanium sapphire laser at 4.7 MHz.

Munaka, T., Kanai, M., Abe, H., Fujiyama, Y., Sakamoto, T., Mahara, A., Yamay-oshi, A., Nakanishi, H., Shoji, S., Murakami, A., In situ cell monitoring on a microchip using time-resolved fluorescence anisotropy analysis. Micro Total Analysis Systems 2003, Proceedings 7th pTAS Symposium, Squaw Valley, CA, Oct. 5-9, 2003, 283-286. 

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

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