Polarization of the Fluorescence

Weber G (1952) Polarization of the fluorescence of macromolecules II. Fluorescent conjugates of ovalbumin and bovine serum albumin. Biochem J 51 155-167... 

F. Weigert Discovery of the polarization of the fluorescence emitted by dye solutions... 

Weber G. (1953) Rotational Brownian Motions and Polarization of the Fluorescence of Solutions, Adv. Protein Chem. 8, 415-459. 

G. Weber, Rotational Brownian motion and polarization of the fluorescence of solutions,... 

Rotational Brownian Motion and Polarization of the Fluorescence of Solutions Gregorio Weber... 

If the molecules are placed in a magnetic field, the different spatial orientations correspond to different Zeeman levels and the selective saturation explained above leads to a nonthermal population of the Zeeman niveaus. By exposing the sample simultaneously to an rf field with the proper frequency, magnetic dipole transitions between the Zeeman levels can occur and the thermal population can be restored. This will again increase the polarization of the fluorescence at most up to its presaturation value. The fluorescence polarization is thus used as detector for the rf transitions. 

In Fig. 12 in Ref 25, fluorescence microscopy images of different dye-loaded zeolite L single crystals are shown. Each line consists of three pictures of the same sample, but with different polarizations of the fluorescence observed. In the first one, the total fluorescence of the crystals is shown, and in the others, the fluorescence with the polarization direction indicated by the arrows is displayed. The zeolite was loaded with the following dyes (A) Py+, (B) PyGY", (C) PyB +, (D) POPOP (see Table 1). Most crystals show a typical sandwich structure with fluorescent dyes at the crystal ends and a dark zone in the middle. This situation can be observed when the diffusion of the dyes in the channels has not yet reached its equilibrium situation. It illustrates nicely how the molecules penetrate the crystals via the two openings on each side of the one-dimensional channels. 

FLUORESCENCE MEASUREMENTS OF LIGAND BINDING. In principle, ligand binding may either enhance or quench the intrinsic or extrinsic fluorescence of its macromolecular receptor or it may change the polarization of the fluorescence emission (see below). 

With vertically polarized exciting light, p0 — when p = 0. But when P = n/2, p0 becomes negative and is equal to —1/3. The values are +1/3 and—1/7 for unpolarized radiation. Thus negative polarization appears when 6 is small, i.e. absorption probability is high and the transition moment in emission is perpendicular to that in absorption. These observations provide a suitable method for assigning the polarization directions of transition moments in different absorption bands of a given molecule from polarization of the fluorescence excitation spectra. 

Guest et al. have studied the polarization of the fluorescence that results from the B( E+) when C1CN is photolyzed at 157.6 nm with a F2 laser (158). They find that the absorption dipole is parallel to the C1CN internuclear axis and that the photodissociation process is direct. 

An interesting detection system which needs no separation step and is amenable to automation is the Abbott TDx analyzer. It is based on measurement of increase in polarization of the fluorescence of a small fluorescent labeled antigen when bound by a larger molecular weight antibody. It has been used for detection of contaminants, especially smaller antigens like drugs and steroids in food (76). Its detection range is pg/ml which can be... 

Weber, G. (1952). Polarization of the fluorescence of macromolecules. I. Theory and experimental method. Biochemical Journal, 51,145-155. 

Fluorescent organic compounds have been widely used as molecular-microscopic probes in biophysical studies of the local environment in micelle-forming surfactant solutions, in phospholipid dispersions, and in membranes. It is assumed that the nature of the probe environment is reflected in its emission characteristics [i.e. position and intensity of emission maxima, vibrational fine structure, quantum yields, excited-state lifetime, polarization of the fluorescence) cf [112, 115, 360] for reviews. 

Fig. 3.5. Scanning confocal image of a glycerol thin film with a low concentration of fluorescent dye molecules at 204 K. The polarization of the fluorescence, recorded by two independent detectors, is rendered as the color of the pixels. The three circled single molecules demonstrate visible differences in their tumbhng patterns. The upper one tumbles at the scanning rate (about f s per line scan), while the lower molecule keeps its orientation during a few successive scans. The middle molecule reorients faster...

On binding of an antigen to an antibody there will be a reduction or a restriction in the rotational Brownian motion of the fluorescent label. This will cause considerable polarization of the fluorescence along or perpendicular to the optical axis of the excitation polarizer, depending upon whether the fluorescence transition moment of the molecule is oriented closer to 0 or 90° to the transition moment associated with the absorption band excited. Let us first consider the case where the transition moments for excitation and fluorescence are parallel (or nearly so). 

The polarization of the fluorescence emitted at 90° is determined by measuring the intensities of the vertically polarized fluorescence (J ) and of the horizontally polarized fluorescence (li), i.e., the intensities after the fluorescence has passed through a polarizer with the polarizing axis set at the vertical and horizontal orientations, respectively. The resultant parameter is ihe fluorescence polarization p. 

The results of a photoselection experiment on a dilute solution of PMPrS in a rigid glass consisting of a 7 3 volume ratio of 3MP and isoP at 77°K are shown in Figure 5. The polarization of the fluorescence, excited at four different wavelengths throughout the lowest absorption band, is shown... 

McConnell and co-workers have demonstrated that information about the orientation of molecules in a condensed monolayer phase can be obtained by examining the polarization of the fluorescence. The underlying principle is shown in Fig. 6. The monolayer is assumed to be made up of domains of tilted amphiphiles and, for simplicity, the polarizability tensor of the fluorescent probe bonded to some of the molecules is taken to be perpendicular to the chain axis. If p-polarized light is directed to the surface at an angle with respect to the surface normal, the fluorescence excited in the domains will not be identical. The contrast difference between the domains will be reversed if the horizontal component of the direction of the... 

Measurement of the degree of polarization of the fluorescence from an excited atomic fragment can give more information than f3 about the relative absorption amplitudes for excitation to different dissociative states. The alignment of photofragment emission can give information above the relative phases of the transition moments for photoexcitation of the parent molecule (3 samples only the squared transition moments), due to interference effects (Vigue,... 

Zare, 1982) may be measured if the molecular photoion is produced in a fluorescing excited electronic state. The polarization of the fluorescence determines the alignment of the nascent photoion. The alignment of the J+ photoion rotational level is defined as... 

A detailed study of the predissociation channel (iii) from the C A state in HCN and DCN, which included analysis of the absorption and photofragment fluorescence excitation spectra, and measurements of the relative quantum yields and polarizations of the fluorescence following excitation into different vibronic levels, provides an example of the interrelation between photochemistry and spectroscopyThis was used to help characterise the nature of the excited electronic parent molecular states and provide information on the topography of the potential surfaces over which the predissociation proceeds. The photofragment fluorescence excitation spectra from HCN and DCN are displayed in Figs. 3.4. and 3.4.8 all the major vibronic bands can be attributed to progressions in the C state, and the CN(B - X) fluor-... 

Use of polarized light to excite fluorescence, and measurement of the state of polarization of the emitted light introduce another set of measurable parameters that can characterize structures and dynamics of molecules. The anisotropy of the polarization of fluoresence after excitation by linearly polarized light provides the rotational diffusion coefficient, or rotational correlation time, of the fluorophore. When there is fluorescence energy transfer, analysis of the anisotropy of both donor and acceptor can reveal the relative orientation, and the relative motion. Measurement of fluorescence after excitation by circularly polarized light provides the fluorescence-detected circular dichroism. This measurement characterizes the chiral environment of the ground state of the fluorophore. If the circular polarization of the fluorescence is measured, the circularly polarized luminescence is obtained. This measurement characterizes the chirality of the excited state. 

Excitation with polarized light creates a partial orientation of selectively excited molecules. The relaxation rate of these excited molecules and its dependence on the degree of orientation can be studied either by the time-resolved absorption of polarized light by the excited molecules, or by measuring the polarization of the fluorescence and its time dependence [1513]. 

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