Fluorometry, time-resolved fluorometries

By the total internal reflection condition at the liquid-liquid interface, one can observe interfacial reaction in the evanescent layer, a very thin layer of a ca. 100 nm thickness. Fluorometry is an effective method for a sensitive detection of interfacial species and their dynamics [10]. Time-resolved laser spectrofluorometry is a powerful tool for the elucidation of rapid dynamic phenomena at the interface [11]. Time-resolved total reflection fluorometry can be used for the evaluation of rotational relaxation time and the viscosity of the interface [12]. Laser excitation can produce excited states of adsorbed compound. Thus, the triplet-triplet absorption of interfacial species was observed at the interface [13]. 

In the mechanism of an interfacial catalysis, the structure and reactivity of the interfacial complex is very important, as well as those of the ligand itself. Recently, a powerful technique to measure the dynamic property of the interfacial complex was developed time resolved total reflection fluorometry. This technique was applied for the detection of the interfacial complex of Eu(lII), which was formed at the evanescent region of the interface when bathophenanthroline sulfate (bps) was added to the Eu(lII) with 2-thenoyl-trifuluoroacetone (Htta) extraction system [11]. The experimental observation of the double component luminescence decay profile showed the presence of dinuclear complex at the interface as illustrated in Scheme 5. The lifetime (31 /as) of the dinuclear complex was much shorter than the lifetime (98 /as) for an aqua-Eu(III) ion which has nine co-ordinating water molecules, because of a charge transfer deactivation. 

Karvinen, J., Laitala, V., Makinen, M. L., Mulari, O., Tamminen, J., Hermonen, J., Hurskainen, P. and Hemmila, I. (2004). Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase. Anal. Chem. 76, 1429-1436. 

Diamandis, E.P. (1993) Time-resolved fluorometry in nucleic acid hybridization and Western blotting techniques (Review). Electrophoresis 14, 866-875. 

SANS has been recently used to study problems related to micelle preparation and kinetics, as reported by Bates and coworkers who have used time-resolved SANS to study molecular exchange and micelle equilibration for PEO-PB diblocks in water [71]. The authors have shown that the micellar structures initially formed upon dissolution were completely locked in up to 8 d after preparation. Fluorometry and DLS have also been used to monitor micelle equilibration [72],... 

Hemmila I, Webb S (1997) Time-resolved fluorometry an overview of the labels and core technologies for drug screening applications. Drug Discov Today 2 373-381... 

The most reliable method for the determination of k3 and k i is based on time-resolved experiments. Either pulse fluorometry or phase fluorometry can be used (see Chapter 6). They provide the values of the decay times from which the rate constants k3 and k i are determined from Eqs (4.52) to (4.53) and the ratio ki/k i yields K. ... 

Dr can be determined by time-resolved fluorescence polarization measurements, either by pulse fluorometry from the recorded decays of the polarized components I l and 11, or by phase fluorometry from the variations in the phase shift between J and I as a function of frequency (see Chapter 6). If the excited-state lifetime is unique and determined separately, steady-state anisotropy measurements allow us to determine Dr from the following equation, which results from Eqs (5.10) and (5.41) ... 

Knowledge of the dynamics of excited states is of major importance in understanding photophysical, photochemical and photobiological processes. Two time-resolved techniques, pulse fluorometry and phase-modulation fluorometry, are commonly used to recover the lifetimes, or more generally the parameters characterizing the S-pulse response of a fluorescent sample (i.e. the response to an infinitely short pulse of light expressed as the Dirac function S). 

Time-resolved emission anisotropy measurements are more straightforward in pulse fluorometry. 

Time-resolved spectra are more easily recorded in pulse fluorometry. 

There are two widely used methods for measuring fluorescence lifetimes, the time-domain and frequency-domain or phase-modulation methods. The basic principles of time-domain fluorometry are described in Chapter 1, Vol.l of this series(34) and those of frequency-domain in Chapter 5, Vol. 1 of this series.<35) Good accounts of time-resolved measurements using these methods are also given elsewhere/36,37) It is common to represent intensity decays of varying complexity in terms of the multiexponential model... 

The operation and application of streak cameras in fluorescence lifetime spectroscopy has been reviewed previously (see, e.g., Refs. 91 and 92). Streak cameras are useful in 2-D time-resolved imaging applications such as microscopy or multiwavelength array fluorometry. The operating principle is based on converting an optical pulse into a photoelectron pulse and spatially dispersing the electron image on a phosphor by means of a synchronized deflection voltage across two plates. 

D. J. S. Birch, K. Suhling, A. S. Holmes, A. D. Dutch and R. E. Imhof, Array fluorometry the theory of the statistical multiplexing of single-photon timing, in Time-Resolved Laser Spectroscopy in Biochemistry II, (J. R. Lakowicz, ed.), Proc. SPIE 1204, 26-34 (1990). 

T. Lovgren, I. Hemmila, K. Pettersson and P. Halonen, Time-resolved fluorometry in immunoassay, in Alternative Immunoassays (W. P. Collins, ed.) pp. 203-217, John Wiley Sons, New York (1985). 

The elucidation of the intramolecular dynamics of tryptophan residues became possible due to anisotropy studies with nanosecond time resolution. Two approaches have been taken direct observation of the anisotropy kinetics on the nanosecond time scale using time-resolved(28) or frequency-domain fluorometry, and studies of steady-state anisotropy for xFvarying within wide ranges (lifetime-resolved anisotropy). The latter approach involves the application of collisional quenchers, oxygen(29,71) or acrylamide.(30) The shortening of xF by the quencher decreases the mean time available for rotations of aromatic groups prior to emission. 

J. R. Lakowicz, H. Cherek, and B. P. Maliwal, Time-resolved fluorescence anisotropies of diphenylhexatriene and perylene in solvents and lipid bilayers obtained from multifrequency phase-modulation fluorometry, Biochemistry 24, 376-383 (1985). 

Braunwalder, A.F., Yarwood, D.R., Sills, M.A., and Lipson, K.E., Measurement of the protein tyrosine kinase activity of c-SRC using time-resolved fluorometry of europium chelates, Anal. Biochem., 238, 159, 1996. 

Nonisotopic receptor assay for benzodiazepine drugs using time-resolved fluorometry. Anal. Chem. 1995, 67, 2655-2658. 

Perkin Elmer Life Sciences Applications of Time-resolved Fluorometry with the DELFIA Method , 2003. http //lifesciences.perkinelmer.com. 

Hukkanen, V. Rehn, T. Kajander, R. Sjoroos, M. Waris, M. Time-resolved fluorometry PCR assay for rapid detection of herpes simplex virus in cerebrospinal fluid. J. Clin. Microbiol. 2000, 33(9), 3214-3218. 

Nurmi, J. Kiviniemi, M. Kujanpaa, M. Sjoroos, M. Ilonen, J. Lovgren, T. High-throughput genetic analysis using time-resolved fluorometry and closed-tube detection. Anal. Biochem. 2001, 299(2), 211-217. 

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