Time-resolved fluorescence polarization

Dr can be determined by time-resolved fluorescence polarization measurements, either by pulse fluorometry from the recorded decays of the polarized components I l and 11, or by phase fluorometry from the variations in the phase shift between J and I as a function of frequency (see Chapter 6). If the excited-state lifetime is unique and determined separately, steady-state anisotropy measurements allow us to determine Dr from the following equation, which results from Eqs (5.10) and (5.41) ... 

The choice of method depends on the system to be investigated. The methods of intermolecular quenching and intermolecular excimer formation are not recommended for probing fluidity of microheterogeneous media because of possible perturbation of the translational diffusion process. The methods of intramolecular excimer formation and molecular rotors are convenient and rapid, but the time-resolved fluorescence polarization technique provides much more detailed information, including the order of an anisotropic medium. 

This relation shows that the rotational correlation time is uncoupled from the excited-state lifetime, in contrast to classical steady-state or time-resolved fluorescence polarization measurements (see Chapter 5). The important consequence is the possibility of observing slow rotations with fluorophores of short lifetime. This is the case for biological macromolecules labeled with fluorophores (e.g. rhodamine) whose lifetime is of a few nanoseconds. 

The broad field of nucleic acid structure and dynamics has undergone remarkable development during the past decade. Especially in regard to dynamics, modem fluorescence methods have yielded some of the most important advances. This chapter concerns primarily the application of time-resolved fluorescence techniques to study the dynamics of nucleic acid/dye complexes, and the inferences regarding rotational mobilities, deformation potentials, and alternate structures of nucleic acids that follow from such experiments. Emphasis is mainly on the use of time-resolved fluorescence polarization anisotropy (FPA), although results obtained using other techniques are also noted. This chapter is devoted mainly to free DNAs and tRNAs, but DNAs in nucleosomes, chromatin, viruses, and sperm are also briefly discussed. 

Time-resolved fluorescence polarization measurements for 1 and 2 in n-heptane showed that after pulsed excitation, the initial value of the fluorescence anisotropy is about 0.25. This... 

Circular polarization of luminiscence, stopped-flow fluorescence, fluorescence-monitored chemical relaxation, the evaluation of relative orientation by polarized excitation energy transfer, time-resolved fluorescent polarization ( nanosecond polarization ), and other new techniques have become valuable means for studying protein structures, their interactions and structural changes in relation to various treatments (e.g. denaturation). New fluorescent probes and quenchers have enabled the research field to expand from isolated proteins to more complicated systems such as membranes, muscle and nerve components and other subcellular structures (see also 7.3). 

The changes have been used to provide information about the enviromnent of the fluorescent probe and to follow changes in conformation of the macromolecule. In other work the study of the fluorescence polarization properties of the attached probe under steady state illumination and the application of Perrin s equation enable calcu-latnn of the rotary Brownian motion of the polymer. This technique has been extended by Jablonski and Wahl to the use of time-resolved fluorescence polarization measurements to calculate rotational relaxation times of molecules These experiments are discussed fiilly in the fdlowing section of this review. 

Fluorescence Polarization Studies of PMA and PAA. Time-resolved fluorescence polarization measurements are potentially a powerful means for studying molecular mobility. The fluorescence anisotropy function r(t) may be generated by monitoring the decay of vertically (Iv(t)) and horizontally (Ijj(t)) polarized components of emission following excitation by vertically polarized light pulses (Equation 1). 

L.A. Kelly, J.G. Trunk, J.C. Sutherland, Time-resolved fluorescence polarization measurements for entire emission spectra with a resisitive-anode, single-photon-counting detector The fluoreseence omnilizer, Rev. Sci. Instrum. 68, 2279-2286 (1997)... 

Time-resolved fluorescence polarization As described by Eq. (10), the anisotropy of spherical particles in a homogeneous isotropic medium decays exponentially. Anisotropy decays, however, can be more complex. The three most important origins for non-monoexponential decays are described in the following ... 

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