Formal fluorescence measurements

The electronic excited state is inherently unstable and can decay back to the ground state in various ways, some of which involve (re-)emission of a photon, which leads to luminescence phenomena (fluorescence, phosphorescence, and chemiluminescence) (22). Some biologic molecules are naturally fluorescent, and phosphorescence is a common property of many marine and other organisms. (Fluorescence is photon emission caused by an electronic transition to ground state from an excited singlet state and is usually quite rapid. Phosphorescence is a much longer-lived process that involves formally forbidden transitions from electronic triplet states of a molecule.) Fluorescence measurement techniques can be extremely sensitive, and the use of fluorescent probes or dyes is now widespread in biomolecular analysis. For example, the large increase in fluorescence... 

The use of atomic fluorescence for the determination of mercury was first reported by Thomson and Reynolds in 1971 [2]. Since then, several authors [3-6] have described enhancements to the technique that have reduced formal instrument detection limits (IDL) for the fluorescence technique to the 1-10 ng/1 range. Knox et al. [7] report on the use of atomic fluorescence detection limit for mercury to less than 1 ng/1. A European standard EN 13506 was published in 2001. This uses vapor generation coupled to direct atomic fluorescence measurement. The most recent version of the US EPA standard 1631 utilizes an additional gold amalgamation step. The amalgamation provides the potential for an additional order of sensitivity but also requires considerable attention to detail and cleanliness to avoid contamination. 

The advent of water-soluble polymers with well-defined hydrophobic centers, such as dodecyl or nonylphenyl side chains (or even pyrene groups), has continued to formalize the concept of a relationship between the hydrophobicity of a polymer and its reactivity. As mentioned earlier, reactivity in a series of alkyl-substituted polyacrylate polymers was shown, in the fluorescence measurements of Magny et al. (90,114), to correlate with the number and chain length of the substituent groups, as was the case for alkyl-substituted maleic anhydride copolymers (115). It is easy to understand that hydrophobicity introduced in this way can lead to interaction through the formation of mixed micelles with the surfactant. (See Fig. 22.) Indeed, as we have pointed out, the interaction affinity in some cases can be so large as to overcome unfavorable electrostatic forces. 

Recently, a formalism has been developed to determine the second and the fourth order parameters of films using polarized total internal reflection fluorescence (TIRF) [71]. Similarly to IR-ATR spectroscopy (Section 4), the experiment makes use of p- and s-polarized excitation, but the fluorescence emission (analyzed either in p- or s-direction) is detected normal to the substrate. Two approaches are developed based on the measurements of two intensity ratios. In the first one, the S angle has to be known experimentally or theoretically, and the order parameters (P2) and (P4) can be determined. In the second one, the order parameter (R ) is obtained by another technique, for instance IR-ATR spectroscopy, which allows deducing the order parameter (P4) and (cos2<5). 

The lifetime of the singlet excited state (the fluorescence lifetime TF) is of the order of picoseconds to 100 nanoseconds (10—12 - 10-7 seconds) and can now be measured accurately using pulsed laser excitation methods and other techniques. Since the radiative transition from the lowest triplet state to the ground state is formally forbidden by selection rules, the phosphorescence lifetimes can be longer, of the order of seconds. 

Several formal and informal intercomparisons of nitric acid measurement techniques have been carried out (43-46) these intercomparisons involve a multitude of techniques. The in situ measurement of this species has proven difficult because it very rapidly absorbs on any inlet surfaces and because it is involved in reversible solid-vapor equilibria with aerosol nitrate species. These equilibria can be disturbed by the sampling process these disturbances lead to negative or positive errors in the determination of the ambient vapor-phase concentration. The intercomparisons found differences of the order of a factor of 2 generally, and up to at least a factor of 5 at levels below 0.2 ppbv. These studies clearly indicate that the intercompared techniques do not allow the unequivocal determination of nitric acid in the atmosphere. A laser-photolysis, fragment-fluorescence method (47) and an active chemical ionization, mass spectrometric technique (48) were recently reported for this species. These approaches may provide more definite specificity for HN03. Challenges clearly remain in the measurement of this species. 

FDCD measurements, and a basic theoretical formalism for this technique, were first reported by Turner, Tinoco and Maestre in 1974 [5]. In this experiment one uses the selectivity and sensitivity of luminescence measurements to probe the local chiral environment of fluorescent chromophores. The ultimate goal in many applications of FDCD is to relate the observed differential fluorescence signal to the conventional CD measurement. In certain multi-component absorbing systems this procedure may be difficult. This technique is sometimes applied to systems for which CD measurements are impossible or very difficult. FDCD, like CPL and other polarization sensitive techniques, is not immune to troublesome background and noise problems, and these will be discussed in Section 3. The only detailed discussion of the applicability of FDCD measurements, and other characteristics of the technique has been presented by Turner in 1978 [6]. In this chapter we will also list some of the more recent applications of FDCD. 

Figure 6. Time-resolved fluorescence of 4>OH state of py ranine as measured when trapped in the aqueous layers of multilamellar liposome made of DPPC plus cholesterol at 1 1 ratio. The fluorescence signal, recorded over 20 ns, is presented on a logarithmic amplitude scale. The overlaid curves are theoretically reconstructed dynamics, calculated according to Agmons formalism (19-21) and modified to account for the geometry depicted in Figure 4. The lowermost curve was calculated for d w = 30 A. In the other curves, dw decreases by equal decrements of 2.5 A to = 20 A (upper curve). Even at the first decrement the predicted curve deviates from the margin determined by the experimental noise in the time range of t 8 ps, where the signal is still 5% of the maximal...

It should be noted that quantitative measurements of FRET efficiency are primarily useful for distance measurements however, there are many experiments in which distance information is not the main objective, e.g., binding isotherms. In such cases, the transition from Emm to (or vice versa) can provide important quaUtative and quantitative information. Changes in acceptor fluorescence intensity, donor fluorescence intensity, or the ratio between the two (Ead/Fda) can be used similarly, without formal calculation of FRET efficiency, when distance information is not the objective. Specific examples where quantitative measurement of FRET efficiency is both important and unimportant are provided later in this entry. 

For example, in the case of pyrene (the most widely used fluorescent dye) the measured parameter is /1//3, the ratio of intensity of the first (372 nm) and third (385 nm) fluorescence peaks for pyrene aldehyde it is the wavelength of maximum absorption (36,37). In the presence of interacting polymer the plot is displaced to lower surfactant concentration this implies that a surfactant aggregation process, formally akin to micelli-... 

The above properties and phenomena can be assessed with great sensitivity and precision by the measurement of rotational diffusion usually based upon the combined use of polarized excitation and deactivation processes. The faster motions alluded to above are particularly well adapted to the techniques of nuclear magnetic relaxation and fluorescence depolarization, the formalisms for which are extensively documented (references 2 and 3, respectively, and citations therein other chapters in this volume). Optical anisotropy decay measurements with longer time resolution have been very effective in studies of biological and model membrane systems (reviewed in 4-6). 

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