Fluorescence polarization anisotropy polarizer alignment

To measine fluorescence polarization, or anisotropy, a sample is excited with polarized light and the emission is measured through a polarizer aligned either parallel or perpendicular to the excitation polarization (Fig. 1.17). If emission occurs from the same state that is generated by excitation, and the excited molecule does not rotate between these two events, then the fluorescence polarized parallel to... 

Homo-FRET is a useful tool to study the interactions in living cells that can be detected by the decrease in anisotropy [106, 107]. Since commonly the donor and acceptor dipoles are not perfectly aligned in space, the energy transfer results in depolarization of acceptor emission. Imaging in polarized light can be provided both in confocal and time-resolved microscopies. However, a decrease of steady-state anisotropy can be observed not only due to homo-FRET, but also due to rotation of the fluorescence emitter. The only possibility of discriminating them in an unknown system is to use the variation of excitation wavelength and apply the... 

Steady-State Anisotropy Following continuous excitation with vertically polarized light, a distribution of fluorophores whose transition vectors for the absorption process are vertically aligned will be photoselected, creating an excited state population, which possesses a degree of anisotropy (r) or optical order, in an otherwise isotropic distribution of fluorophores. Measurement of the intensity of fluorescence, via an emission polarizer in planes parallel (z n) and perpendicular (zx) to the vertical plane allows estimation of r from... 

Anistropy measurements are based on the photose-lective excitation of fluorophores by plane-polarized light. In an isotropic medium, the fluorophores are randomly oriented. Upon excitation with polarized light, those fluorophores whose absorption transition dipole is aligned parallel to the electric vector of the excitation, will be preferentially excited. If the molecule rotates and tumbles out of this plane during the excited state, light is emitted in a different plane from the excitation light. The intensity of the emitted light can be monitored in vertical and horizontal planes and thus, fluorescence anisotropy (r) and polarization ( ) are defined by ... 

A number of macromolecular diffusion and conformational properties can be studied using fluorescence anisotropy, fluorescence correlation spectroscopy (ECS), and fluorescence recovery after photobleaching (FRAP). These techniques most commonly are applied to proteins labelled with highly fluorescent probes, but can exploit intrinsic fluorescence in some instances. In fluorescence anisotropy studies, polarized light is used to selectively excite molecules whose transition dipole moments are aligned with the electric field vector. Steady-state... 

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