Kinetics fluorescence intensity - time

Fig. 22.1. (A) Enzymatic cycle of cholesterol oxidase which catalyzes the oxidation of cholesterol by oxygen. The enzyme s naturally fluorescent FAD active site is first reduced by a cholesterol substrate molecule, generating a non-fluorescent FADH2, which is then oxidized by oxygen. (B) Structure of FAD, the active site of cholesterol oxidase. (C) A portion of the fluorescence intensity time trace of a single cholesterol oxidase molecule. Each on-off cycle of emission corresponds to an enzymatic turnover. (D) Distribution of emission on-times derived from (C). The solid line is the convolution of two exponential functions with rate constants fci[S] = 2.5 s and fc2 = 15.3 s, reflecting the existence of an intermediate, ES, the enzyme-substrate complex, as shown in the kinetic scheme in the inset. From ref. [15]...

Kinetic Analysis of Fluorescence Intensity - Time Curves... 

The "add-to-memory" signal averaging method currently available to us distorts fluorescence intensity versus time plots when the fluorescence intensity is a non-linear function of incident laser energy and the laser energy varies from shot to shot. For this reason we have not attempted detailed kinetic modelling of the observed fluorescence intensity decay curves recorded at high 532 nm laser fluence. 

Kinetic data for total and nonspecific binding are subtracted to give the specific amount bound at the time of antibody addition, and the slow decline in fluorescence intensity thereafter reflects dissociation of bound peptide (also see Ref. 8 for more details). 

Sanden, T., Persson, G., Thyberg, P., Blom, H. and Widengren, J. (2007). Monitoring kinetics of highly environment sensitive states of fluorescent molecules by modulated excitation and time-averaged fluorescence intensity recording. Anal. Chem. 79, 3330-41. 

The kinetics of complex formation with Zn2+ can be followed by monitoring the change in the fluorescence intensity [ 17g]. In the case of 1, the change in the fluorescence intensity with time indicates a biphasic kinetics with the incorporation rate constants k1 = 4.9 x 105 M 1 s 1 followed by a first order process... 

The time evolution of the fluorescence intensity of the monomer M and the excimer E following a d-pulse excitation can be obtained from the differential equations expressing the evolution of the species. These equations are written according to the kinetic in Scheme 4.5 where kM and kE are reciprocals of the excited-state lifetimes of the monomer and the excimer, respectively, and ki and k i are the rate constants for the excimer formation and dissociation processes, respectively. Note that this scheme is equivalent to Scheme 4.3 where (MQ) = (MM) = E and in which the formation of products is ignored. 

G(t) decays with correlation time because the fluctuation is more and more uncorrelated as the temporal separation increases. The rate and shape of the temporal decay of G(t) depend on the transport and/or kinetic processes that are responsible for fluctuations in fluorescence intensity. Analysis of G(z) thus yields information on translational diffusion, flow, rotational mobility and chemical kinetics. When translational diffusion is the cause of the fluctuations, the phenomenon depends on the excitation volume, which in turn depends on the objective magnification. The larger the volume, the longer the diffusion time, i.e. the residence time of the fluorophore in the excitation volume. On the contrary, the fluctuations are not volume-dependent in the case of chemical processes or rotational diffusion (Figure 11.10). Chemical reactions can be studied only when the involved fluorescent species have different fluorescence quantum yields. 

The dynamics will be deduced from the time dependencies of the fluorescence intensities of both precursor and product states using advanced kinetic theories. These are presented in Section IV. One of the... 

A kinetic-fluorimetric method for the determination of choline and acetylcholine by oxidation with cerium (IV) was reported by Lunar et al. [47]. To sample solutions containing 0.017-1.OmM choline and/or acetylcholine were successively added 6M H2S04 (5mL) and 7.1 mM Ce(IV) solution (0.35 mL), the mixture diluted to 10 mL with water, and the solution heated to 80° C for 2 min. A portion of the solution was transferred to a cell maintained at 20 0.1°C, and after 1 min the Ce(III) fluorescence intensity was measured at 360 nm (excitation at 260 nm) as a function of time. 

Flash photolysis of misonidazole, metronidazole, and nitrobenzothiazoles has been carried out in [1369-1371], Laser flash-photolysis (355 nm) allows to determine relatively stable anion-radicals of misonidazole and metronidazole in aqueous solutions [1370], Solvated electrons have been formed at harder irradiation, the result of which interaction with nitroimidazole molecules is generation of their radical anions [1372], The authors [1372] have also found that fluorescence intensity of metronidazole is about 20 times more than that of misonidazole in same conditions. Photochromic properties of benzothiazole derivatives containing nitro and methyl groups in the ortho positions with respect to each other were studied by flash photolysis [1371], The application of the thermodynamic approach to predict the kinetic stability of formed nitronic acids is limited owing to specific intramolecular interactions. The lifetime of photoinduced nitronic acid anions tends to increase with rise in the chemical shift of the methyl protons. The rate constants photoinduced nitronic acids and their anions increase as the CH3C-CN02 bond becomes longer [1371],... 

Figure 2. a) The decrease in tryptophan fluorescence intensity after a 0 "C to 20 "C temperature jump is shown. The fluorescence intensity can be fit with a 5 ns time constant giving the time resolution of the instrument, b) Monitoring the tryptophan fluorescence intensity shows that the temperature remains elevated for several milliseconds. Relaxation kinetics can be measured over a time range of 5 ns to 2 ms with this instrument. 

Fig. 4. Percent uptake of different iiposomai formuiations conjugated to Rtiodamine by human DCs measured through flow cytometry. Ceiis were incubated with Rhodamine-MSP-1, g-ioaded iiposomai formuiations and anaiyzed at different time intervals (0,15,30,60,120,180, and 360 min). The kinetics of uptake has been presented with mean fluorescence intensity (MFI) vs. counts by FACS analysis and percentage uptake at various time inten/als. Uptake of formulation on a per cell basis was quantified as fluorescence intensity per cell. Percentage of positive cells was determined as proportion ot cells with fluorescence intensity higher than 99% of cells of the control sample (cells incubated with unconjugated rhodamine alone). Flow cytometric analysis revealed that the percentage of rhodamine-positive DCs increased rapidly and reached a plateau after 16 h of incubation (means of three independent experiments). A steady increase in the uptake percentage (%) was recorded and a maximum cell-associated fluorescence was observed at 16 h for OPM-coated cationic liposomes. Flow cytometric analysis of DCs revealed that plain liposomes did not significantly enhance the antigen uptake by DCs compared with the uptake recorded for mannan-coated liposomes...

Figure 2.8. Kinetic trace of AO.D. at 590 nm of TMB + during the pulse radiolysis followed by the consecutive irradiation of a 532-nm laser pulse. Fluorescence intensity (open circle) as a function of the delay time of the 532-nm laser pulse relative to the electron pulse is superimposed on the decay curve.

Thus, the question of central concern raised in our contribution has been the macroscopic formulation of EET and its relation to the experimental observable of excimer fluorescence in a time-resolved experiment. EET has been discussed, hers, as a dispersive, i.e., time-depen-dent process in deterministic monomer-excimer models which had been the subject of a detailed kinetic analysis in recent work (3 8, 4.S.). With the use of rate function k(t) (Equation 4) it is natural to yield typical non-exponential intensity-time profiles, either in form of an asymptotic approach (Equations 5,6), or in closed form analytical solutions (Equations 7,8). The physios emer-... 

---