Fluorescence Polarization or Anisotropy

Anisotropy measurements are widely used in biochemistry and are even used for clinical immunoassays. One reason for this use is the ease with which these absolute values can be measured and compared between laboratories. 

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The versatility of luminescence goes beyond intensity-, wavelength- and kinetic-based measurements. Fluorescence polarization (or anisotropy) is an additional parameter still largely unexplored for optical sensing yet widely used in Biochemistry to study the interaction of proteins, the microfluidity of cell membranes and in fluorescence immunoassays. Although only a few optosensors based on luminescence polarization measurements can be found in the literature, elegant devices have recently been reported to measure chemical parameters such as pFI or O2 even with the bare eye41. 

Anotiier ratiometric mediod is based on die measurement of fluorescence polarization or anisotropy. In this case the analyte causes a change in the polarization of the label. Anisotropy measurements are frequently used in competitive immunoassays, in which the actual analyte displaces labeled analyte that is bound to specific antibody. This results in a decrease in the anisotropy. Anisotropy values are calculated using the ratio of polarized intensity measurements. The use of an intensity ratio makes the anisotropy measurements independent of fluorophm c(mi-centration as long as the measuremeDts are not distorted by autofluoresccnce or pow signal-to-noise ratio. Polarization immunoassays are discussed in Section 19.9X). 

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... 

In principle, this would allow the fluorescence polarization or anisotropy to be determined with just two spectral acquisitions Vy and Hy. However, in practice, it is vital to correct for the differing efficiencies of the emission monochromator and detector towards vertically and horizontally polarized light, which may be substantial. Consequently, for isotropic samples. Equation [1] can be amended to... 

Polarization effects in monochromators and other optical components can present significant difficulties in the overall operation and calibration of fluorimeter systems. At the same time, the introduction of polarizing elements such as polarizing filters or Glan-Thompson or Glan-Taylor polarizing optics are essential to the measurement of fluorescence polarization or anisotropy. 

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. 

The timescale of fluorescence emission is comparable to that of rotational diffusion of proteins and the timescale of segmental motions of protein domains or individual amino acid residues. The polarization or anisotropy of the emission provides a measure of these processes. Suppose a sample is excited with vertically polarized light (Fig. 11), and that the sample is viscous so that the fluorophores do not rotate during the lifetime of the excited state. Then the emission is polarized, usually also in the vertical direction. This polarization occurs because the polarized excitation selectively excites those fluorophores in the isotropic solution whose absorption... 

Huorescence anisotropy measurements and their applications are covered in Chapter 3. Plane polarized exciting light can be obtained by placing a Polaroid sheet or a Clan Taylor prism in front of the sample holder. The polarization or anisotropy of the fluorescence of the sample is then determined by placing another polarizer in front of EmM. The fluorescence that is polarized parallel, J, and perpendicular, to the excitation polarization direction is then measured. 

Another development is the combination of FLIM with other modalities, such as polarization or anisotropy measmements. This has resulted in the measurement of spatially resolved anisotropy decay measmements in microscopy [41, 55-57]. Basically, conventional FLIM measmements are combined with methods to determine the polarization state of the fluorescence after using polarized excitation light. This is done by measming fluorescent intensity and... 

Let us consider tire case of a donor-acceptor pair where tire acceptor, after capturing excitation from tire donor, can emit a photon of fluorescence. If tire excitation light is linearly polarized, tire acceptor emission generally has a different polarization. Common quantitative expressions of tliis effect are tire anisotropy of fluorescence, r, or tire degree of polarization,... 

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

Situation with H-bonding also demands to take into account the fact that alcohols have ability to form various associates or even clusters at normal conditions. The most efficient method for determination of inhomogeneity in the excited states is fluorescence polarization measurements. These methods also frequently applied for studying of solvent viscosity, they may be provided in two variants steady state and time-resolved. Relations for time-resolved and steady state fluorescence anisotropy may be given as [1, 2, 75] ... 

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) ... 

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. 

The emitted light is detected along y through a polarizer oriented either along z (Fz) or along x (Fx). In fluorescence polarization studies with continuous excitation (steady-state experiments), the emission anisotropy r and the emission polarization p are defined in eqs 8a and 8b. 

Anisotropy. Light emitted from excited molecules immediately after absorption is always partially polarized, whether or not the exciting beam consists of plane polarized light. When light polarized in a vertical plane is used for excitation, part of the emitted light (of intensity lv) will have its electric vector parallel to that of the exciting light. The remainder of intensity /, will be polarized in a horizontal plane. The polarization P of the emitted radiation is defined by Eq. 23-19 and the anisotropy R by Eq. 23-20. After excitation by a laser pulse both the fluorescence and its anisotropy decay with time and can be measured. The decay of R (but not of P) can usually be described as the sum... 

In Eq. (14.1), I is fluorescent intensity the subscript letters, V for vertical and H for horizontal, represent the polarization direction of the two polarizers on the excitation and emission light path, respectively and the ratio, ZHV/IhH) calibrates for the difference in the emission channel s sensitivity towards vertical and horizontal polarized components. Anisotropy, r, can be measured by either L-format or T-format. In the L-format, all four fluorescence intensities, Zw, h11, f iv. and ZHh> are measured using a single channel of a photodetector so that each intensity needs to be measured separately. If the fluorimeter has two emission channels then anisotropy can also be measured in a T-format, which allows fluorescence intensities pairs, Ivv//Vi i or If iv // ii i, to be measured simultaneously via the two emission channels. Thus, measurements in the T-format are faster than in the L-format. 

FP is an alternative readout principle for endopeptidase activity assays. FP or anisotropy measurements allow the detection of changes in the rotational correlation time of particles. These differences in the rotational correlation (or relaxation) time are related to different masses of particles. The experimental determination of steady-state fluorescence anisotropy requires the linear polarization of the light used for the excitation of the probe as well as linear polarization of the emitted fluorescence. Based on data of an appropriate experiment, the fluorescence anisotropy can be calculated as ... 

Fluorescence Polarization under continuous excitation are presently developed. But these experiments need the a priori choice of a model of motion to be interpreted, and such models do not exist so far for the local dynamics in bulk polymers. This limitation is very troublesome, since experiments carried out on polymers in solution have shown that varying the choice of the model used in data treatment could lead to important discrepancies in the derived correlation times or activation energies. In the following, we will show how Fluorescence Anisotropy Decay may help to overcome this difficulty, and we will give some examples of original information that can be obtained using this technique in conjunction with the powerful synchrotron light source. 

The following sections give an overview and some examples of the fluorescence detection methods currently used in HTS fluorescence intensity (FI) fluorescence polarization (FP) or fluorescence anisotropy (FA) fluorescence resonance energy transfer (FRET) Hfetime-based measurements (TRF and FLT) fluorescence corre-... 

The theory for rotational diffusion of ellipsoids, and measurements by fluorescence polarization, can be traced to the classic reports by F. Perrin. Since these seminal reports, the theory has been modified to include a description of expected anisotropy decays. Hiis theory has been summarized in several reviews.For a rigid ellipsoid with three unequal axes, it is now agreed that the anisotropy decays with five correlation times. The correlation times depend on the three rotational diffiision coefficients, and the amplitudes depend on the orientation of the absorption and emission transition moments widiin the fluoroi iore and/or ellipsoid. While the the( predicts five correlation times, it is known diat two pairs of correlation times will be very close in magniOide, so that in practice only three correlation times are expect for a nonsf oical molecule. ... 

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