Fluorescence intensity total

While a laser beam can be used for traditional absorption spectroscopy by measuring / and 7q, the strength of laser spectroscopy lies in more specialized experiments which often do not lend themselves to such measurements. Other techniques are connnonly used to detect the absorption of light from the laser beam. A coimnon one is to observe fluorescence excited by the laser. The total fluorescence produced is nonnally proportional to the amount of light absorbed. It can be used as a measurement of concentration to detect species present in extremely small amounts. Or a measurement of the fluorescence intensity as the laser frequency is scaimed can give an absorption spectrum. This may allow much higher resolution than is easily obtained with a traditional absorption spectrometer. In other experiments the fluorescence may be dispersed and its spectrum detennined with a traditional spectrometer. In suitable cases this could be the emission from a single electronic-vibrational-rotational level of a molecule and the experimenter can study how the spectrum varies with level. 

Electronic transitions in molecules in supersonic jets may be investigated by intersecting the jet with a tunable dye laser in the region of molecular flow and observing the total fluorescence intensity. As the laser is tuned across the absorption band system a fluorescence excitation spectrum results which strongly resembles the absorption spectrum. The spectrum... 

Agglomerated impurities, such as particles or droplet residues, do not participate in the interference phenomenon leading to total reflection their fluorescence intensity is independent of the angle of incidence below the critical angle, and drops by a factor of 2 if the critical angle is surpassed due to the disappearance of the reflected component in the exciting beam nonreflecting impurities and residues). 

Since then, TXRE has become the standard tool for surface and subsurface microanalysis [4.7-4.11]. In 1983 Becker reported the angular dependence of X-ray fluorescence intensities in the range of total reflection [4.12]. Recent demands have set the pace of further development in the field of TXRE - improved detection limits [4.13] in combination with subtle surface preparation techniques [4.14, 4.15], analyte concentrations extended even to ultratraces (pg) of light elements, e. g. A1 [4.16], spe-dation of different chemical states [4.17], and novel optical arrangements [4.18] and X-ray sources [4.19, 4.20]. 

Fig. 4.14. Fluorescence intensity from layers buried in a thick substrate. The dependence of intensity on the glancing angle was calculated for layers of different thickness but with a constant analyte area density. Silicon was assumed as substrate and Mo-Ka X-rays as primary beam. Total reflection occurs in the region below 0.1°. Without total reflection, the dashed horizontal line would be valid throughout [4.21].

Quantitative aspects. The total fluorescence intensity, F, is given by the equation F = Ia(f> where Ia is the intensity of light absorption and 4>f the quantum efficiency of fluorescence. Since 70 = 7a + 7t where 70 = intensity of incident light and 7t = intensity of transmitted light, then... 

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). 

Both WDXRF and EDXRF lend themselves admirably to quantitative analysis, since there is a relationship between the wavelength or energy of a characteristic X-ray photon and the atomic number of the element from which the characteristic emission line occurs. The fluorescence intensity of a given element is proportional to the weight fraction. Emitted fluorescence radiation is partly absorbed by the matrix, depending on the total mass absorption coefficient ... 

Here Fmin is the fluorescence intensity without binding and /,max is the intensity when the sensor molecules are totally occupied. Kd is the dissociation constant. The differences in intensities in the numerator and denominator allow compensating for the background signal, and the obtained ratio can be calibrated in target concentration. But since F, Fmin and Fmax are expressed in relative units, they have to be determined in the same test and in exactly the same experimental conditions. This requires proper calibration, which is difficult and often not possible. 

We can also count the total number of donor-emitted photons, or measure the corresponding analog intensity, in the presence and absence of energy transfer. From these intensities we can calculate the efficiency of energy transfer. The fluorescence intensity of the donor is proportional to the rate constant through the fluorescence pathway divided by the sum of the rates of leaving the excited state by all pathways. That is,... 

In addition, one needs to know Io, the total (unquenched) fluorescence intensity corresponding to the At slices from which I is determined. The ratio Io /I is related to a specific concentration of solvent (quencher) through independent knowledge of the fluorescence quenching process. 

Si - Si Annihilation and Ablation Mechanism. The Si - Si annihilation process is responsible to laser ablation, which was supported by the following experiment. Total fluorescence intensity and the relative intensity of excimer emissions (-15 72 ns gate width) were plotted against the fluence in Figure 3. It is interesting that the relative contribution of excimers showed a similar change to that of total fluorescence intensity. This indicates that the Si - Si annihilation has an important role in the primary processes of laser ablation phenomena, since the relative contribution of excimers is determined by the degree of Si -Si annihilation, and the suppressed fluorescence intensity corresponds to the enhanced ablation. 

The total fluorescence intensity saturated around a few hundreds of mJ/cm2 which corresponds to the irradiation condition where the new plasma-like emission was observed. Above this value fluorescence intensity decreased, which is accompanied with the recovery of the relative intensity of excimer emissions. This means that a quite efficient deactivation channel of excitation intensity opens in this energy range, and the contribution of Si -Si annihilation is depressed. This suggests that fragmentation reactions to diatomic radicals are not induced by the annihilation process. Multi-photon absorption processes via the Si states and chemical intermediates should be involved, although no direct experimental result has as yet been obtained. 

In the expression of the polarization ratio, the denominator represents the fluorescence intensity in the direction of observation, whereas in the formula giving the emission anisotropy, the denominator represents the total fluorescence intensity. In a few situations (e.g. the study of radiative transfer) the polarization ratio is to be preferred, but in most cases, the use of emission anisotropy leads to simpler relations (see below). 

Note that in none of the directions Ox, Oy, Oz, is the observed fluorescence intensity proportional to the total fluorescence intensity. They are respectively Iy + I f I + I f and 2I . It will be shown in Chapter 6 (see... 

Appendix) how a signal proportional to the total fluorescence intensity can be measured by using excitation and/or emission polarizers at appropriate angles. 

The components ly and Ih, vertically and horizontally polarized respectively, are such that Jz = Jy = Ix, Iy = IH (Figure 5.3). The total fluorescence intensity is then 2fy + Ih- The polarization ratio and the emission anisotropy are given by... 

Following an infinitely short pulse of light, the total fluorescence intensity at time t is I(t) = J (t) + 2 I (t), and the instantaneous emission anisotropy at that time is... 

On continuous illumination (i.e. when the incident light intensity is constant), the measured anisotropy is called steady-state anisotropy r. Using the general definition of an averaged quantity, with the total normalized fluorescence intensity as the probability law, we obtain... 

This equation shows that, at time t, each anisotropy term is weighted by a factor that depends on the relative contribution to the total fluorescence intensity at that time. This is surprising at first sight, but simply results from the definition used for the emission anisotropy, which is based on the practical measurement of the overall ly and I components. A noticeable consequence is that the emission anisotropy of a mixture may not decay monotonously, depending of the values of r, and Ti for each species. Thus, r(t) should be viewed as an apparent or a technical anisotropy because it does not reflect the overall orientation relaxation after photoselection, as in the case of a single population of fluorophores. 

The total fluorescence intensity at time t is obtained by summing over all molecules emitting at that time. Because there is no phase relation between the elementary emissions, the contributions of each molecule to the intensity components along Ox, Oy and Oz are proportional to the square of its transition moment components along each axis. Summation over all molecules leads to the following expressions for the fluorescence intensity components ... 

Let Ix, Iy and Iz be the intensity components of the fluorescence, respectively (Figure 6.3). If no polarizer is placed between the sample and the emission monochromator, the light intensity viewed by the monochromator is Iz + Iy, which is not proportional to the total fluorescence intensity (Ix + Iy + Iz). Moreover, the transmission efficiency of the monochromator depends on the polarization of the incident light and is thus not the same for Iz and Iy. To get a response proportional to the total fluorescence intensity, independently of the fluorescence polarization, polarizers must be used under magic angle conditions (see appendix, p. 196) a polarizer is introduced between the excitation monochromator and the sample and... 

Distortion of the fluorescence response measured by the detection system (monochromator + detector) arises when the emitted fluorescence is partially polarized. As explained in the Appendix, a response proportional to the total fluorescence intensity can be observed by using two polarizers an excitation polarizer in the vertical position, and an emission polarizer set at the magic angle (54.7°) with respect to the vertical, or vice versa (see the configurations in Figure 6.3). 

The total fluorescence intensity is thus again equal to f +2ij. This equality is therefore valid whatever the value of 9. 

The ratiometric measurements are preferable because the ratio of the fluorescence intensities at two wavelengths is in fact independent of the total concentration of the dye, photobleaching, fluctuations of the source intensity, sensitivity of the instrument, etc. The characteristics of some fluorescent pH indicators allowing ratiometric measurements are given in Table 10.1. 

Note that a2/b2 represents the ratio of the absorbances or fluorescent intensities of the free ligand and the complex at the wavelength X2 a,2/bi = Yo(22)/Yiim( 2)-Equation (B.22) can be used for the determination of Ky, only if the concentration in free cation [M] can be approximated to the total concentration cM. 

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