Depolarization principles

Detection of Bromine Vapor. Bromine vapor in air can be monitored by using an oxidant monitor instmment that sounds an alarm when a certain level is reached. An oxidant monitor operates on an amperometric principle. The bromine oxidizes potassium iodide in solution, producing an electrical output by depolarizing one sensor electrode. Detector tubes, usefiil for determining the level of respiratory protection required, contain (9-toluidine that produces a yellow-orange stain when reacted with bromine. These tubes and sample pumps are available through safety supply companies (54). The usefiil concentration range is 0.2—30 ppm. 

Any factor that affects the size or shape of a molecule, the hindered movement of a fluorophore within a molecule, or the energy transfer within the molecule will affect the measured depolarization of its fluorescence emission. Therefore, the conformation of humic fractions in solution can be studied as a function of pH, ionic strength, temperature, and other factors by depolarization measurements. The principle of the method is that excitation of fluorescent samples with polarized light stimulates... 

Accurate experimental determinations require that not only the handedness of the produced light, but its exact degree of polarization, are known. The theoretical performance of an undulator may be in practice be degraded by magnetic defects, and the optical beam can be further depolarized by reflections along the beamline. Again, the dephasing on optical elements can in principle be... 

Because there is no phase relation between the light emitted by different molecules, fluorescence can be considered as the result of three independent sources of light polarized along three perpendicular axis Ox, Oy, Oz without any phase relation between them. Ix, Iy, Iz are the intensities of these sources, and the total intensity is I = Ix + Iy + Iz. The values of the intensity components depend on the polarization of the incident light and on the depolarization processes. Application of the Curie symmetry principle (an effect cannot be more dissymmetric than the... 

Lipari G. and Szabo A. (1980) Effect of Vibrational Motion on Fluorescence Depolarization and Nuclear Magnetic Resonance Relaxation in Macromolecules and Membranes, Biophys. J. 30, 489—506. Steiner R. F. (1991) Fluorescence Anisotropy Theory and Applications, in Lakowicz J. R. (Ed.), Topics in Fluorescence Spectroscopy, Vol. 2, Principles, Plenum Press, New York, pp. 127-176. 

One would still like to examine the effect of ethidium on the torsional rigidity and dynamics at high binding ratios. One would also like to test the Forster theory for excitation transfer between bound ethidium molecules, since it has been questioned/65- This is possible in principle by deconvoluting the effects of depolarization by excitation transfer on the FPA, as will be shown subsequently. DLS also provides crucial information on this same question. 

A detailed discussion of the statistical thermodynamic aspects of thermally stimulated dielectric relaxation is not provided here. It should suffice to state that kinetics of most of the processes are again complicated and that the phenomenological kinetic theories used to described thermally stimulated currents make use of assumptions that, being necessary to simplify the formalism, may not always be justified. Just as in the general case, TSL and TSC, the spectroscopic information may in principle be available from the measurement of thermally stimulated depolarization current (TSDC). However, it is frequently impossible to extract it unambiguously from such experiments. 

Lastly, we consider the diffusive contribution to the signal. Since this portion of the signal arises from molecular reorientation, it should be completely depolarized unless these diffusive reorientational dynamics also have a significant DID component. The orientational decay will be made up of exponential components, the number of which depends on the molecular symmetry and the relationship between the principal axes of the diffusion and polarizability tensors of the molecules (8). If these tensors share no axes, the orientational decay will be composed of a sum of five exponentials. If the tensors share one axis, the decay will be composed of three exponentials. If the tensors share all three axes, the decay will be composed of two exponentials. If the molecule is further a symmetric top, then reorientation about the axis of symmetry cannot be observed, and the decay will be composed of a single exponential. In principle, considerably more information is available when the principal axes of the diffusion and polarizability tensors are not shared however, in practice it is virtually impossible to find a unique fit to the sum of five exponentials, some of which may have very small amplitudes. In the remainder of this chapter we will therefore concentrate on symmetric-top liquids. 

FP assays are known to be susceptible to artifacts (Turek-Etienne and Small, 2003). In principle, the assays are ratiometric and should normalize for variations in total excitation energy applied as would occur with inner filter effects, and newer generations of red-shifted fluorophores should help to eliminate interference (Vedvik et al., 2004). However, introducing a test compound with fluorescent or absorbent properties at 5 or 10 pM with the typically sub-micromolar concentrations of fluorophores in an FP assay can significantly skew the measurements. For example, if the compounds are insoluble, they can scatter and depolarize light. A concentration-dependent effect on an FP assay could result from an increase in the amount of insoluble compound. 

The technique of fluorescence depolarization uses in principle fluorescent dyes covalently bound to proteins. The label is excited by polarized light the fluorescence is also polarized to a degree that is inversely related to the amount of Brownian motion occurring during the interval between absorption and emission of the light. The information about the amount of motion helps to form an idea on the size and shape of the protein being investigated. 

---