Fluorescence timing, absolute

Absolute fluorescence timing, 226f Absorbance spectra, for albumin solution, 71f Absorbance spectrometry multiwavelength measurement, 66f wide dynamic range array, 57-74 Acridine... 

Despite the nearly identical absorption spectra of both polymers, the emission quantum yield of poly(dA) is nearly three times that of poly(A), as can be seen from the unsealed emission spectra (Fig. 1) measured from polymer solutions having identical absorbance at the excitation wavelength of 280 lim. The absolute fluorescence quantum yield of poly(A) has previously been reported as 3 x 10 [16]. Interestingly, the situation is reversed at 77 K, and more fluorescence was reported from the ribo- than from the deoxyribopolymer [15]. The... 

Figure 1. The absolute flourescence timing. Rise of fluorescence of M center in NaF. Curve a shows 30 shots ofexcitation pulse scattered by a thin (300 pm) crystal, and Curve b shows 30 shots of fluorescence rise. Calculated curves show solution to differential equations assuming response times ofr-Ops, 3ps, and 12ps. These results imply a rapid relaxation process with r < / ps.

It is often experimentally convenient to use an analytical method that provides an instrumental signal that is proportional to concentration, rather than providing an absolute concentration, and such methods readily yield the ratio clc°. Solution absorbance, fluorescence intensity, and conductance are examples of this type of instrument response. The requirements are that the reactants and products both give a signal that is directly proportional to their concentrations and that there be an experimentally usable change in the observed property as the reactants are transformed into the products. We take absorption spectroscopy as an example, so that Beer s law is the functional relationship between absorbance and concentration. Let A be the reactant and Z the product. We then require that Ea ez, where e signifies a molar absorptivity. As initial conditions (t = 0) we set Ca = ca and cz = 0. The mass balance relationship Eq. (2-47) relates Ca and cz, where c is the product concentration at infinity time, that is, when the reaction is essentially complete. 

In general, luminescence measurements are relative rather than absolute, since the Instrument characteristics and sample properties that determine the fluorescence Intensities are often not well defined. Absolute luminescence measurements are difficult to perform and require time and Instrumentation not available In most laboratories. Thus, luminescence measurements rely heavily on standards to determine Instrument responses and parameters, the chemical composition of samples, and the characteristics of chemical systems. To... 

Fluorescence techniques are the most sensitive. With proper dye selection and proper experimental conditions, the absolute sensitivity may reach the limit of single molecules. This feature is especially needed if the target exists in trace amounts. High sensitivity may allow avoiding time-consuming and costly target-enrichment steps. 

Collection of multiple data sets for each time span, with frequent alternation of the polarization, is an essential feature of our protocol. This provides some protection against the effects of drifts in laser power, photomultiplier quantum yield, and absolute calibration of the TAC, photochemical decomposition of the dye, and any other long-term processes that may alter the measured fluorescence response curves. Separate analysis of each data set is necessary to provide an indication of the uncertainty in run-to-run reproducibility and to detect and delete the rare spurious data set. 

The system provides a very sensitive means of detection levels of 10 picograms absolute are measurable with the continuous (permanent) trapping system. A further advantage is that the software calculates the analytical results directly in concentration in the unit volume of sample introduced. However, it should he stressed that the level of mercury measured is an absolute quantity and while the detection Hmit is of the order of 10 picograms, this quantity can be contained in any volume of gas. In addition, the fact that the mercury both absorbs and fluoresces to provide a measurement which can be measured with a specific retention time provides more positive evidence of the presence of mercury. 

The run-to-run reproducibility of the profile shape of the FOCS fluorescence-intensity signal is good however, the reproducibility of the absolute intensity values is unsatisfactory. The run-to-run variations in the fluorescence intensities are caused by the differences in resin thickness at the small area "viewed" by the optrode. In addition, substantial resin flow takes place during cure, causing the resin thickness to vary as a function of cure time. However, since this variation in resin thickness mi t be reproducible from run-to-run (if other cure parameters remain unchanged), it may be possible to develop a suitable... 

Absolute measurements of the 2PA cross section of chromophores are experimentally very demanding and difficult to implement for routine studies of new materials. This is mainly due to the fact that the 2PA excitation rate in a sample does not depend simply on the square of the average intensity, (f(r, t)), but on the average of the square of the intensity, (f(r, t) ) (r and t are the special and temporal coordinate) [63,64,78]. These two quantities are not the same if the laser piflse has substructure in space and time [85,86]. Thus, absolute 2PA determinations that rely on the measurements of signals proportional to the excitation rate, such as the 2P-induced fluorescence... 

Figure 13. Time-resolved decay-associated spectra of the UV (protein) fluorescence components i, rp)i - 4 of Pr phytochrome (124 kDa) and of the red-light adapted Pr + Pfr mixture obtained by global analysis. The dashed line corresponds to the stationary fluorescence spectrum obtained by A c = 295 nm (cf. Figure 11). The amplitudes of the two sets of spectra can be compared on an absolute basis (Holzwarth et al. [108]).

Concentration Profiles. The relative fluorescence intensity profiles for OH, S2, SH, SO, and SO2 were converted to absolute number densities according to the method already outlined. Resulting concentration profiles for a rich, sulfur bearing flame are exhibited in Figure 17. H-atom densities were calculated from the measured OH concentrations and H2 and H2O equilibrium values for each flame according to Equation 6. Similar balanced radical reactions were used to calculate H2S and S concentrations 6). Although sulfur was added as H2S to this hydrogen rich flame, the dominant sulfur product at early times in the post flame gas is S02 ... 

The method proposed allows direct absolute measurement of local concentration at the instant of the laser pulse in a low pressure flame. We believe that this method could be applied to higher pressure flames by the use of ultrashort duration laser pulses with the new mode locked dye laser technique. But until the detector technology allows such short time resolutions we think that collisional lifetimes studies must be pursued to obtain more precise evaluation of the fluorescence efficiency, and to have a better understanding of the redistribution phenomena involved in optical pumping. For this purpose we are now studying the decay of resolved fluorescence lines. 

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