Fluorescence Polarisation Methods

The availability of MIP microparticles through this synthetic method has also stimulated the development of analytical techniques that make use of them as sensing elements. Apart from competitive radioassays [30] and immunoassays [32], which were already performed with ground bulk polymers, the small, regular size of the beads prepared by dispersion/precipitation polymerisation enables their use in CEC [45, 46], scintillation proximity assays [35], fluorescent polarisation assays [47], and chemiluminescence imaging [48]. 

Figure 11.23. Energy level diagram and observed transitions for SrF in its X2 + state, showing the 19F hyperfine splitting [43], observed because of the higher resolution obtained with the microwave/optical polarisation method. This diagram may be compared with figure 11.21, appropriate for the earlier lower resolution studies employing conventional fluorescence detection.

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

In hair analysis, three methodically different lA with different kinds of labeling are used radioimmunoassay (RIA), which is the most common lA in hair analysis, enzymeimmunoassay (EIA), and fluorescence polarisation immunoassay (FPIA). In hair analysis, lA have the same advantages and disadvantages as compared to their use in urinalysis. They are fast, easy to handle, and can be automated. Their use can save time and expenses when a great number of negative samples has to be expected. 

Production of fluorescent LCs by dissolving fluorescent dyes in host materials has been an active area of smdy for many years. This is because it is the simplest method for producing fluorescent LCs and also because the absorption and emission profiles of the dyes can be altered by application of an electric field. Examples of potential applications include polarised organic lasers, but the main problem with the use of fluorescent dyes in guest-host systems is the limited solubility of the... 

Figure 11.1. (a) Principles of the microwave/optical double resonance method, (b) Change of polarisation of fluorescent light resulting from AM= 1 radiofrequency transitions. 

The polarisation of the beam can be measured by a number of techniques. Probably the most precise method is fluorescent monitoring. Information on the polarisation of the ground state can be obtained from the polarisation of the fluorescent light (Fischer and Hertel, 1982). Simi-... 

The principle of the method is similar to that of the infrared method except that the polymer is illuminated by polarised radiation of a suitable wavelength to induce fluorescence and the emitted radiation is examined using an analyser. Information is thus obtained about molecular orientation with respect to two directions, the transmission directions of the polarizer and analyser. 

In Table 3 the orientation information which can be obtained from these various structural techniques is summarised. This table also shows the part of the molecular structure which is being characterised, and some of the theoretical and experimental limitations of each method. A further technique, that of polarised fluorescence has been added. This technique is exactly analogous in its orientation aspects to Raman spectroscopy. The distinction between the two techniques lies in the fact that in the Raman effect, the lifetime of the process is of the order of the vibrational period ( 10 s) whereas fluorescence occurs after much longer occupancy of the transition state ( 10 s). 

The first treatment of the theory of the intensities and polarisation effects to be expected in the Raman scattering from an oriented polymer sample appears to be that given by Cornell and Koenig." This treatment must be regarded at best as a very rough approximation, since the tensor nature of the effect is not taken into account properly. Snyder has given a correct account of the theory for rather special distributions of orientations of the Raman scatterers but his work concentrates on the information that can be obtained about the Raman tensors if the orientation distribution is known. The only treatment that has considered how much information can in principle be obtained about the distribution of orientations and what measurements are necessary to obtain it is that of Bower and Bower and Purvis. " In this treatment the similarities and differences between the theories of the fluorescence and Raman methods are apparent and an account of it follows. 

The choice of fluorescent probe depends on a variety of factors. It has already been pointed out that what is determined directly is information which characterises the distribution of orientations of the fluorescent molecules. The ideal experiment would be one in which the polymer molecules themselves contained fluorescent groups. Stein has considered the theory of the fluorescence method specifically for a uniaxially oriented fluorescent rubber but no experiments to study orientation have been reported for such a system. Nishijima et al have, however, made some qualitative observations on the polarisation of the fluorescent light from polyvinylchloride films which had been first stretched and then irradiated with light of wavelength 185 nm to produce fluorescent polyene segments. 

PPP films can also be prepared by electropolymerisation imder either reductive or oxidative conditions, but the EL properties have been found to be highly dependent on the polymerisation conditions [51]. A study of the PL efficiency of PPP thin films of varying chain length concluded that for highly ordered PPP films a chain length of 25-30 units was optimal [52]. Oriented films of PPP have been prepared by a friction deposition method and found to show highly polarised fluorescence [53]. 

Although the relation between fluorescence depolarisation and rotational Brownian motion was first identified by Perrin and the development of the theoretical background of the time-resolved fluorescence depolarization experiments was made by Jablonski use of the technique was limited until the advent of improved fluorescence decay time measurements some fifteen years i. An alternative, related technique, involving excitation using a continuous polarised light source, provides only the time average of the correlation function (Eq. 18) and as such, is less useful than the time resolved method. Other disadvantages are that the natural decay time of the chromophore must be determined from a sqrarate experiment and it is necessary to alter the viscosity, and/or temperature of the medium, often withun-... 

---