Fluorescent technique polarization

In addition to wavelength and time-resolved fluorescence techniques, polarization fluorescence can yield important information about an analyte." This is especially true when differentiating between chiral compounds. Combining, for example, a fluorescently tagged antibody immunoassay with polarization detection allows for very sensitive detection limits of chiral enantiomers. Laser-induced fluorescence polarization (LIFP) has been used to detect concentrations as low as 0.9 nM of an antibody-boimd cyclosporine A (an immunosuppressive drug) in human blood. A conventional single channel fluorescence detector can be easily modified to perform such measurements, simply by adding the appropriate polarization filters. 

It should be noted that no emission from the zwitterionic form of the proton-transferred tautomer was observed from any of the benzotriazoles studied in the present work. This implies that non-radiative relaxation processes from the excited state of this species are very efficient in all of the solvent and polymer environments studied. Thus no information is available on the effect of the medium polarity on the room-temperature photophysics of the zwitterionic form using fluorescence techniques. 

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. 

In addition to fluorescence intensity and polarization, fluorescence spectroscopy also includes measurement of the lifetime of the excited state. Recent improvements in the design of fluorescence instrumentation for measuring fluorescence lifetime have permitted additional applications of fluorescence techniques to immunoassays. Fluorescence lifetime measurement can be performed by either phase-resolved or time-resolved fluorescence spectroscopy. 

Until recently, previous studies for continuous monitoring of hepatic function with ICG utilized the absorption mode. However, new studies demonstrate that the highly sensitive fluorescence technique can equally be used [148-150]. In addition to high sensitivity, in-depth analysis of the emission, excitation and polarization properties of fluorescence spectroscopy furnishes additional functional information about the dye molecule. In this system, the fluorescence profile emanating from the clearance of injected biocompatible dye is monitored with a small photodetector. Fig. 8 shows the in vivo fluorescence detection apparatus developed for continuous monitoring of organ functions [147,148]. 

Morr, C. V., Van Winkle, Q. and Gould, I. A. 1962. Application of polarization of fluorescence technique to protein studies. III. The interaction of x-casein and (3-lacto-globulin. J. Dairy Sci. 45, 823-826. 

Fluorescence techniques have been used with great success in the study of PEO-fe-PSt micelles [64]. In this study, the effect of polymer concentration on the fluorescence of pyrene present in water at saturation was studied. Three features of the absorption and emission spectra change when micellization occurs. First, the low-energy band of the (S2-So) transition is shifted from 332.5 to 338 nm. Second, the lifetime of the pyrene fluorescence decay increases from 200 to ca. 350 ns, accompanied by a corresponding increase in the fluorescence quantum yield. Third, the vibrational fine structure changes, as the transfer of pyrene from a polar environment to a nonpolar one suppresses the permissibility of the symmetry-forbidden (0,0) band. 

Infrared dichroism is one of numerous methods used to characterize molecular orientation. The degree of anisotropy of the strained pol3rmers may also be accurately characterized by other techniques such as X-ray diffraction, birefringence, sonic modulus, polarized fluorescence and polarized Raman spectroscopy [2]. These techniques directly probe the orientational behavior of macromolecular chains at a molecular level, in contrast to the macroscopic information provided by mechanical measurements. 

Multiple parameters can be measured for fluorescence photons count rate (or intensity), wavelength (X), polarization (p), arrival time (to), time delay after excitation (td), and location x,y on an imaging detector). These parameters carry information about the fluorophore that includes the nature of its environment, its interactions with other molecules, and its motions. Fluorescence techniques that exploit each of these parameters are described below. 

The fluorescence depolarization technique for mobility and ordering is based on the fact that the probability of absorption and emission is directional. Light polarized along a certain axis will preferably excite molecules oriented with their transition dipole moment in the same direction. The probability varies with cos 0, where 0 is the angle between the transition dipole moment and the electric field vector of the light. Emission of a photon obeys the same cos 0 (28) rule. That means that a molecule oriented with its transition dipole moment along the Z-axis will be likely to emit a photon with the same polarization. In the depolarization technique, polarizers are used to quantify the intensity of the parallel (ly) and perpendicular (Ij.) components to the original direction of polarization. 

Appraisals of the optical properties of lipid solutions and dispersions will provide information on concentrations, aggregation and stability, phase transitions, densities, and repeating structuresJ Measurements of refractive index, scattered light intensity (polarized and depolarized), and birefringence are relatively easy laboratory methods on which certain product specifications may be based. Also, fluorescent techniques can readily provide information on lipid movements and transfer of lipid between particles. ... 

However, more recently, the application of fluorescence techniques has attracted attention. Anufrieva et al. applied the polarized luminescence method to the stoichiometric hydrogen bond... 

Among nonisotopic techniques, fluorescence (both intrinsic and extrinsic) offers a convenient mode of detection, and the sensitivity of some fluorescent labels is comparable to that of radiolabeled iodine. Recent innovations include the use of polarized light for excitation, such that the degree of polarization of the emission as well as its intensity can provide information about the concentration and size-related behavior (e.g., rotational diffusion) of the fluorescent-labeled molecule. One disadvantage of steady-state fluorescence techniques is that many analytical samples either autofluoresce or quench the fluorescence of the substance of interest. A recent development that circumvents this problem utilizes long-lived fluorophores such as the lanthanide metal ions as labels. Detection is time resolved and data are collected after the decay of spurious or otherwise unwanted fluorescence, i.e., after 100-200 psec. 

Studies on the Structure of Synthetic Polypeptides in Solution by Polarization of Fluorescence Techniques... 

HPhe polarization of fluorescence technique employing dye-macromolecule x conjugates is a sensitive hydrodynamic method for studying the structure and interactions of proteins 19, 20, 41, 54, 65) and synthetic polypeptides 26, 30, 31, 49)- The relationship describing the dependence of polarization of fluorescence upon the Brownian rotational diffusion of the macromolecule was developed by Perrin 50) and extended by Weber 65) in the form of the equations (for excitation with natural light) ... 

These studies begin to provide a systematic basis for the detailed interpretation of polarization of fluorescence measurements in structural terms as well as hydrodynamic data about a wide range of linear and cross-linked synthetic polypeptides. The further development of such studies will provide a firm basis for applying polarization of fluorescence techniques to the study of the structure of native proteins in solution. 

The helix-coil transition can be demonstrated by polarization of fluorescence techniques, and the results may be compared with spectroscopic measurements to correlate the change in the hydrodynamic properties of the molecule with the change of its conformational structure. It is clear that the hydrodynamic and conformational changes do not necessarily parallel one another. Ion adsorption, breaks in the helix, and changes in helical type can occur without being reflected in the optical rotation parameters. 

---