Intensity and polarization of fluorescence

We will now analyze in greater detail the connection between the polarization moments of an excited molecular state and the characteristics of the emitted light. 

A = J —J f. Here the Dyakonov tensor 1 q characterizes the polarization of the registered light. In deriving Eq. (2.24) the properties of the 17-functions were used, as previously in the case of Eq. (2.21) and according to (A.13) and (B.4). It may thus be seen from (2.24) that only the multipole moments bpQ of rank K 2 have any direct effect on the intensity and polarization of molecular fluorescence. This latter assertion also holds in the case where multipole moments of rank higher than K = 2 are created in the excited state (6) in the case of absorption of sufficiently intensive light. 

There are two methods of experimental determination of the magni-tude of the multipoles bPQ and of restoration of the shape of the spatial distribution Pb 6, p) of the angular momenta of the ensemble. One may measure either the intensity of radiation in a certain direction, but with different polarization characteristics (by changing the polarization devices in front of the light detector), or one may measure the intensity of radiation in different directions. The former method is technically more convenient and is therefore applied more frequently. 

Both quantities are interconnected by simple expressions, namely, 

It is now possible to express the fluorescence intensities in Eqs. (2.25) and (2.26) through the multipole moments bpQ of the excited level  

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The aspects of fluorescence spectroscopy that may have value for solving problems in food science and technology have been summarized in a review article by Strasburg Ludescher (Strasburg et al., 1995). In this review article, the techniques described, which depend on the measurement of the intensity, energy and polarization of fluorescence emission, have been illustrated by examples taken from the food science and related literature. 

However, quantitative analysis is more complicated. We [4.10,11] have recently given theoretical arguments to show that the angular distribution, intensity, and polarization of the Raman or fluorescent scattering signal will not only depend upon the number of active molecules but also upon the particle size, shape, refractive index, internal structure, and the distribution of the active molecules within the particle. This will be illustrated in this chapter by representative calculations. 

Solvent Influence. Solvent nature has been found to influence absorption spectra, but fluorescence is substantiaHy less sensitive (9,58). Sensitivity to solvent media is one of the main characteristics of unsymmetrical dyes, especiaHy the merocyanines (59). Some dyes manifest positive solvatochromic effects (60) the band maximum is bathochromicaHy shifted as solvent polarity increases. Other dyes, eg, highly unsymmetrical ones, exhibit negative solvatochromicity, and the absorption band is blue-shifted on passing from nonpolar to highly polar solvent (59). In addition, solvents can lead to changes in intensity and shape of spectral bands (58). 

In addition to fluorescence intensity and polarization, fluorescence spectroscopy also includes measurement of the lifetime of the excited state. Recent improvements in the design of fluorescence instrumentation for measuring fluorescence lifetime have permitted additional applications of fluorescence techniques to immunoassays. Fluorescence lifetime measurement can be performed by either phase-resolved or time-resolved fluorescence spectroscopy. 

The medium in which a species is dissolved or on which it is adsorbed may exert considerable influence on the intensity and wavelength of the fluorescence. Polar materials such as alcohols or esters frequently increase the intensity of the fluorescence relative to non-polar hydrocarbon solvents. The solvent environment often prevents or inhibits intersystem crossing to a triplet state in favour of excitation to a singlet state and fluorescence, while in many cases the opposite is true. The dielectric constant of solvents has been shown to influence the fluorescence intensity and wavelength maxima of some compounds [33,34]. Fig.2.9 shows the effect of solvent dielectric constant on the fluorescence intensity of DNS-phenol, while Table 2.4 shows the corresponding effect on the fluorescence wavelength [34]. For DNS-phenol, solvents of low dielectric constants result in the most intense fluorescence and shift the wavelength maxima to lower values. 

Let us follow in time the intensity /p (t) of fluorescence linearly polarized at an angle linearly polarized excitation and with the geometry... 

Most fluorescent substances have rigid structures associated with fused aromatic rings and their fluorescence intensity is practically independent of the viscosity of the environment (4). On the other hand, their rotational movement as a whole will, of course, depend upon the local environment and since such rotations sweep out a larger volume than would be the case for auramine O, for example (i.e., larger V in eq. 2), so that larger domains in the polymeric system can be studied. Any fluorescent system will exhibit a polarization of fluorescence by virtue of the fact that the fluorescent molecules are anisotropic in regard both to emission and to absorption. This anisotropy can be described by fixed axes within the molecule, namely the dichroic axis of the molecule and the emission... 

Dapoxyl dyes are highly environment-sensitive fluorescent compounds. Their fluorescence in water is very low, but induces a large change of fluorescence intensity, Stokes shifts, and extinction coefficients depending on pH and polarity of solvents. 

ESPT has been identified as the main nonradiative pathway in the excited state of ethidium bromide (EB, I), a popular DNA probe [46a]. In aqueous solution, on addition of DNA, EB intercalates in the double helix of DNA [44-46]. This causes a nearly 11-fold increase in the emission intensity and lifetime of EB. The emission quantum yield and lifetime of EB are very similar in methanol and glycerol, whose viscosities differ by a factor of 2000 [46a]. Thus, the fluorescence enhancement of EB on intercalation is not due to high local viscosity. Emission intensity of EB is low in highly polar, protic solvents, such as alcohol... 

Think of the excited state electrons as objects on a shelf. The electrons will have a natural tendency to fall off the shelf at a rate ( intrinsic) that will depend on the specific molecular structure. If, in addition, the shelf is being rattled by the continual Brownian motion bombardment of surrounding molecules and groups, then the electrons may be displaced by environmental interactions. The rate at which this occurs ( environmental) will depend on the frequency of molecular collisions (temperature) and on the size and polarity of the colliding species. For example, more polar molecules in the surrounding solvent will tend to interact more readily with the excited state electrons owing to electrostatic forces. So we might anticipate that fluorescence intensities will be reduced by an increase in temperature or by transfer to a more polar solvent. 

Hemoproteins are also interesting to investigate because of the efficient energy transfer from tryptophans to heme. Although fluorescence parameters such as intensity, lifetime and polarization of tryptophans in hemoproteins are weak, they still can be measured. Energy transfer rate between a tryptophan and heme depends on the dipole -dipole orientation and the distance that separates the two chromophores. Thus, in a certain way, energy transfer will be affected by the internal dynamics of the protein. Anyway, residual motions will always affect energy transfer between a donor and an acceptor, independently of the chemical nature of the two molecules. 

The electrochemical cell is mounted on a moveable holder, allowing measurements at different angles and polarizations of the incident light. Unfortunately, the number of atoms absorbing radiation is very small (a monolayer of atoms or even less), thus simple absorption measurements like the ones that can be achieved with bulk samples are impossible. Instead, the measurement of Auger electrons (possible only under UHV conditions) or of fluorescence intensity is possible. 

Although there is nothing incorrect about the notion of polarization, its use should be discouraged. Anisotropy is preferred because most theoretical expressions are considerably simpler when expressed in terms of this parameter, an observation first made by Alexander Jablofiski. As an example of this simplification, consider a mixture of fiuorophores, each with polarization Pi and a fractional fluorescence intensity/. The polarization of this mixture (F) is given by ... 

Figure Fluorescence intensity (o) and polarization ( ) of TNS aqueous solution with 0.1 % amylose as a function of the TNS concentration at 25 G,...

Figure 4 shows the dependences of the fluorescence intensity and polarization for TNS-amylose system on the concentration of TNS. It is seen, in contrast to the fluorescence intensity, that the fluorescence polarization of the solution is constant over the range of TNS concentration examined. This fact suggests that the molecular chain of aunylose does not undergo any great conformational change by the formation of complex with TNS. 

The effect of temperature on the fluorescence intensity and polarization for TNS-amylose system is shown in Figure 5- The fluorescence intensity decreases with increasing temperature. Table II shows the fluorescence lifetimes for TNS-amylose system that were determined at 25, 40, and 55 The fluorescence intensity is found to decrease proportionally with decreasing the average lifetime. Thus, the decrease in fluorescence intensity can be ascribed to the change in microenvironmental properties at binding sites of TNS, but not to the decreased amount of binding TNS. 

Figure 6 shows the pH dependences of the fluorescence intensity and polarization for TNS-amylose system. The fluorescence intensity maintains a steady level until the pH reaches 11, and then increases slightly until it reaches a peak at pH 12 before it decreases again at higher pH, The fluorescence polarization is also seen to remain constant until pH reaches 11, and then decrease steeply until pH 12,... 

It has been shown that amylose in an aqueous solution exhibits considerable changes in intrinsic viscosity and optical rotation in the region of pH between 11 and I3, and this behavior has been interpreted in terms of the conformational change caused by increasing negative charges on the polymer chain (12-1 ). We can conclude that the changes in fluorescence intensity and polarization observed here are due to the same conformational transition of amylose. 

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