Fluorescence measurement , correcting

Rush et al.51 first described the effect of thermally induced conformational changes on migration behavior of a-lactalbumin. A sigmoidal dependency of the viscosity-corrected mobility on temperature was observed. Transition temperature also agreed closely with that determined by intrinsic fluorescence measurements. 

B. Fanget, O. Devos, and M. Draye, Correction of Inner Filter Effect in Mirror Coating Cells for Trace Level Fluorescence Measurements, Anal. Chem. 2003, 75, 2790. 

Fluorescence measurement of the Trp residues of the protein in the absence of TNS was performed in a total volume equal to 1060 and 1100 for the native and denatured proteins, respectively. Addition of TNS increases the initial volume and so dilutes the protein solution. Therefore, in order to see the real effect of TNS addition on the protein fluorescence, we should correct the fluorescence intensities for the dilution effect. In the present experiments, we can see that the volume of TNS added to the protein solution is small, and so corrections should have only a minor effect. However, let us do these corrections using the following equation ... 

To avoid reabsorption problems and thus low-emission intensities, the fluorescer concentration should not exceed 10 A/. Typically the fluorescer concentration range is taken between 10 to 10 A/for the double reciprocal plot. Should it be necessary to work at much higher fluorescer concentration, correction for reabsorption is essential. This is readily done by measuring the fluorescer emission intensity as a function of pathlength. From a plot of fluorescer emission intensity vs. path-length one extrapolates /pf at zero pathlength and applies the necessary correction. 

Figure 5. The data processing system with actual time-resolved fluorescence measured for a 5 10 4 M rhodamine B in ethanol (I-mm pathlength) excited by a O.I-mJ, 530-nm laser pulse. The data obtained initiallyfrom the OMA are corrected by computer. In these data, the average time between points (individual OMA channels) is0.8 ps. (Reproduced with permission from Ref. 26. Copyright 1980, North-Holland Publishing Company.)...

Luminescence Measurements. Corrected fluorescence and phosphorescence spectra were obtained using a Hitachi Perkin-Elmer MPF-4 spectrofluorimeter. Fully corrected excitation spectra were obtained also using the newly developed spectrofluorimeter of Cundall, et al. (see Ref. 9, instrumental details to be published). 

This year s group of publications upon the rare-gas halides includes a careful study of the ordering of the B and C states in XeCl, XeBr, and KrCl by observation of the temperature dependences of the C A and B A emissions. " For KrCl, the alphabetic ordering of the states is confirmed, whereas the B state is found to lie above the C for the xenon halides. " The value of Eq—Eb for XeCl, —130 35 cm" differs considerably from that estimated from XeCl fluorescence measurements carried out at room temperature, 5.4 25 cm". The determination of the correct ordering of these states is of some importance, not only for overall modelling of the rare-gas halide laser systems, but also because if the C state lies sufficiently below the B, then lasing on the C A transition becomes possible, as has been shown in the successful operation of the XeF (C - A) laser at 470 nm. Measurements of the polarization of fluorescence from XeF(5) produced by 193 nm photolysis of XeFj has been... 

On the theoretical and spectroscopic side of the subject, one of the more interesting developments during the year has been the reformulation by Colpa on a strict SCF approximation of Hund s rules for predicting the ordering of electronic states. This appears to avoid some of the difficulties recently recognized in the conventional basis of these rules. Calculations on NH and CH8 have led Colbourn to question the normal assumption that electrons in triplet species are more widely separated than in the corresponding singlets. One may also note the important survey of effects of polarization in fluorescence measurements presented by Cehelnik and his co-workers. Correction for such effects may sometimes be necessary. 

Figure 17. The effects of tissue attenuation on fluorescence measurements, (a) graph showing measured fluorescence versus Photofrin concentration in different tissues (after Panjehpour et al., 1993 [12]), (b-d) correction for attenuation by the ratio of fluorescence to diffuse reflectance at two different radial distances (b) principle, (c) prototype instrument, (d) photosensitizer concentration measured in vivo versus known concentration by assay on tissues ex vivo for three different tissue types (after Weersink et al., 1997 [13]).

Since the determination of peak areas, peak detection, and baseline correction are well established methods, the determination of concentration is a smaller problem and results in values with small standard deviations. Nevertheless, TLC is not suitable for automation in its application to photokinetics since the photoactivation of the sample by the chromatographic material is not negligible [108]. The light paths for reflection and fluorescence measurements are given in principle in Fig. 4.27. 

Chlorophyll fluorescence was measured using a pulse amplitude modulation fluorometer (PAM, Heinz Walz, Effeltrich, FRG) as described by [7]. The methods and definitions from [7] were employed to calculate the quenching components qQ, qNP and qTot, which are respectively photochemical quenching, non-photochemical quenching and total quenching of variable fluorescence, all corrected for Fo-quenching (qFo). At each light intensity level, the leaf disc was allowed to achieve steady state of 02-production and fluorescence and then Fs, Fv and Fo were measured. 

Since the majority of materials analyzed using XRF are prepared as solid samples (of infinite thickness), matrix correction procedures must be applied to fluorescence measurements. Several approaches are described in this section, their use depending on both the application and the range of elements to be determined. 

The Compton scatter matrix correction is based on the observation, referred to above, that the intensity of the Compton scatter peak is inversely proportional to the bulk matrix attenuation correction factor. Matrix corrections may then be applied by simply normalizing all fluorescence measurements from a sample to the intensity of the Compton scatter line derived from one of the characteristic fluorescence lines from the X-ray source. This procedure is, however, subject to an important restriction. Corrections are only valid providing no significant absorption edge intervenes between the energy of the Compton scatter peak and the fluorescence line... 

The cac-values obtained from both drop-weight and static fluorescence measurements are shown in Figure 3 and were used in the corrections for the aggregated surfactant concentrations. 

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