Fluorescence, polarization

The term polarization has been used in the previous paragraphs in a generic way, to indicate a specific spatial orientation either of an electromagnetic field or a transition moment. Indeed this word had, when associated to fluorescence a specific meaning being a size alternative to FA. Fluorescence polarization is defined as  

the use of polarized light to excite a solution of fluorescent molecules results in preferential excitation of the molecules that have their transition moments (dipoles) parallel to the direction of the polarized light of excitation. The polarization of their emitted light depends upon how fast the fluorescent probes rotate during the lifetime of its excitation state the faster the rotation, the smaller the polarized signal (P). 

The fluorescent probes usually used in FP possess fluorescent lifetimes shorter than 10 ns. For such probes, significant changes in their polarized signal can be detected in a range of molecular mass between 5 and 40 kDa.  

A large number of homogeneous assays have been developed using FP by inducing a dramatic change in the molecular mass of the moiety carrying the fluorescent 

The ratiometric nature of the polarized signal makes FP independent of the fluorescence intensity and more tolerant to fluorescence quenching and hght scattering than the fluorescence intensity-based measurements.  

However, fluorescence background from chemical compounds or biological media can affect the accuracy of FP. As such fluorescence background mainly arises in the wavelength range between 400 and 550 mn, the use of red dyes emitting above 550 nm can be an effective way to partially overcome this problem.  

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Fig. 7. Fluorescence polarization (FP). (a) The formation of the large FITC—protein A—IgG complex which leads to a net increase in plane polarized light transmitted from the solution. Molecular weights of the protein A-FITC, IgG, and complex are ca 43,000, 150,000, and 343,000, respectively, (b) Detection of IgG by fluorescence polarization immunoassay using A, a laboratory fluorimeter where (O) represents AP = change in polarization, and B, a portable detection unit where (D) is —fiV = change in voltage (27). The field detector proved to be more sensitive than the fluorimeter.

Fluorescence. The fluorescence detection technique is often used in clinical chemistry analyzers for analyte concentrations that are too low for the simpler absorbance method to be appHed. Fluorescence measurements can be categorized into steady-state and dynamic techniques. Included in the former are the conventional simultaneous excitation-emission method and fluorescence polarization. 

Draw ratio X-ray method IR spectroscopy method Absorption Absorption band 898 band 1368 cm cm Optical method Fluorescent polarization method D R S D F method s... 

Inoue, S., et al. (2002). Fluorescence polarization of green fluorescence protein. Proc. Natl. Acad. Sci. USA 99 4272-4277. 

La, S. Y., and Shimomura, O. (1982). Fluorescence polarization study of the Ca2+ -sensitive photoprotein aequorin. FEBS Lett. 143 49-51. 

In order to further extend the utility of fluorescence methods the use of time-resolution methods, fluorescence polarization, and laser techniques should be explored. The addition of other dyes with enhanced fluorescence properties on binding and increased selectivity to various types of nucleic acids will be necessary to further develop more useful analytical methods. 

Figure 4. Continuous analysis by fluorescence polarization of FLPEP binding and dissociation to permeabilized neutrophils. Each curve represents the average of two determinations. The descending curves represent the effect of the addition of receptor antagonist after FLPEP had bound to the receptors. Upper panel, in the absence of GTPyS, receptor antagonist added at t = 60 s lower panel, in the presence of GTPyS, antagonist added at t =...

Lipp, M.M., Lee, K.Y.C., Waring, A., and Zasadzinski, J.A. Fluorescence, polarized fluorescence, and Brewster angle microscopy of palmitic acid and lung surfactant protein B monolayers. Biophys. J. 1997, 72, 2783-2804. 

To perform structural research on a food stuff into which a colorant is incorporated, special properties of fluorescing molecules are exploited fluorescence efficiency, fluorescence lifetime, fluorescence quenching, radiationless energy (Foerster) transfer, stationary or time-dependent fluorescence polarization and depolarization." Generally, if food colorants fluoresce, they allow very sensitive investigations which in most cases cannot be surpassed by other methods. 

Fluorescence polarization anisotropy of a ligand and its complexes with DNA or RNA can be given by the following expression ... 

Parameters Radiometric proximity assays (SPA, Flashplate) Fluorescence polarization (FP) Time- resolved fluorescence (HTRF) Amplified luminescence (ALPHAScreen) Enzyme (p-galactosidase) complementation Electrochemilumines cence... 

Figure 12 Resynthesized library hits identified from the high throughput fluorescence-polarization assay along with their Src SH2 binding data.

Lynch BA, Loiacono KA, Tiong CL, Adams SE, MacNeil IA. A fluorescence polarization based Src-SH2 binding assay. Anal Biochem 1997 247 77-82. 

Unilamellar vesicles PC Polar head/acyl core Fluorescence polarization (DSHA) 33 381... 

Unilamellar vesicles, PC Hydrocarbon core Fluorescence polarization (AS) 2 381... 

Polarization of the Emission. We have sought support for the weakly interacting chain segment model from measurements of room temperature fluorescence polarization (19) on dilute solutions of 1 in 3-methylpentane. An independent preliminary report of similar measurements on a dilute glassy solution at 77K and on a neat polymer has also appeared (21). In the latter case, the analysis is complicated by inter-chain energy transfer. 

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