Emission anisotropy applications

In Chapter 5, devoted to fluorescence polarization, it was shown that information on the rotational motions of a fluorophore can be obtained from emission anisotropy measurements. Application to the evaluation of the fluidity of a medium, or molecular mobility, is presented below. 

Partial orientation is much easier to achieve and generally sufficient to determine the direction of the transition moments, particularly with respect to any axes of symmetry that the molecules may have. Partial orientation can be achieved by photoselection using a linearly polarized excitation source, by application of an electric field or by dissolving the solute in a transparent anisotropic medium (liquid crystals, stretched polymers).182 Photoselection is the basis of emission anisotropy measurements discussed below. It is also used for molecules that can be either generated or destroyed photochemically in a rigid medium such as poly(methyl methacrylate) or glassy solvents at low temperature. Preferential alignments of dipolar molecules that are achievable by electrostatic fields are unfortunately fairly small. 

In another type of application a low molecular fluorescent probe is added to a system containing macromolecules. As would be expected, the rotation of a small species is insensitive to the molecular weight of high polymers, but depends on the "microscopic viscosity" which is a function of free volume. For instance, Nishijima has shown that the microscopic viscosity of liquid paraffin hydrocarbons levels off for molecular weights above 1000 and that the microscopic viscosity of polystyrene containing 10 volume"/ benzene is only 200 times as high as that of benzene (15). Nishijima also showed that the emission anisotropy is a useful index of molecular orientation. Since both the excitation and the emission are anisotropic, the method yields the fourth moment of the distribution function of orientations, while other optical properties (dichroism, birefringence) depend on the second moment (15). 

Fluorescence is a well-observed phenomenon characteristic of many materials and the different forms of their aggregation. Meantime the vast majority of studies on fluorescence have been on small organic molecules in liquid solutions. Parameters of their emission (intensity, lifetime, anisotropy, and positions of excitation and emission spectra) were found to be extremely sensitive to intermolecular interactions [1], which justifies their extensive application in various sensing technologies... 

The elucidation of the intramolecular dynamics of tryptophan residues became possible due to anisotropy studies with nanosecond time resolution. Two approaches have been taken direct observation of the anisotropy kinetics on the nanosecond time scale using time-resolved(28) or frequency-domain fluorometry, and studies of steady-state anisotropy for xFvarying within wide ranges (lifetime-resolved anisotropy). The latter approach involves the application of collisional quenchers, oxygen(29,71) or acrylamide.(30) The shortening of xF by the quencher decreases the mean time available for rotations of aromatic groups prior to emission. 

Summary. X-ray line emissions from ultrashort high-intensity laser-produced plasma were studied in order to clarify the physics of energy transport associated with the generation of ultrashort X-ray pulses for use in various applications. This article reviews two topics. The first is the application of Ka spectroscopy to the study of energy transport in laser-produced plasma. The second topic is the application of X-ray polarization spectroscopy to measurements of the anisotropy of hot electrons generated with ultrashort high-intensity laser pulses. 

The appeal of fluorescence spectroscopy in the study of biomolecular systems lies in the characteristic time scale of the emission process, the sensitivity of the technique, and its ability to accommodate rapid and facile changes in the solvent milieu under conditions corresponding to thermodynamic equilibrium. The time scale of the emission process invites exploitation in two related manners. First, information on hydrodynamic aspects of the system is available from steady-state or time-resolved measurements. Second, detailed information on local dynamic processes within the biomolecular matrix may be derived. Information on hydrodynamic aspects of a macromolecular system may be used to study binding processes, that is, the association of small ligands with macromolecules or macromolecule-macromolecule interactions. In this chapter we focus on the latter applications of polarization or anisotropy data. We shall also try to clarify aspects of this area that our experience has shown to be occasionally misunderstood by initiates. 

Since the PEEM technique is based on the emission of electrons, its application is restricted to pressures below about 10 mbar. Eor higher pressures, two optical methods were adopted ellipsometry for surface imaging (EMSI) and reflection anisotropy microscopy (RAM) [15]. 

Figure 11.19 A schematic illustration of the application of fluorescence anisotropy measurements to study orientation changes in skeletal muscle fibres. A rhodamine-based fluorescent probe is bound to the regulatory light chain (RLC) of the myosin head. Changes in probe orientation directly related to changes in the steady-state orientation of the emission transition dipoles (yellow arrows) of the fluorescent probes in the laboratory frame which are monitored by fluorescence anisotropy. Stretches of the muscle fibre cause the transition dipoles to tilt towards the fibre axis with a corresponding decrease in the fluorescence anisotropy...

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