Direct fluorescence polarization immunoassay.

Direct fluorescence polarization immunoassay of serum cortisol was established. Non-specific binding of fluorescent-labelled cortisol to serum proteins was successively eliminated by sodium dodecyl sulfate. The minimal amount of cortisol detected was 0.1 ng/tube and serum concentration of 1.0 microgram/dl to 100 microgram/dl of cortisol could be measured. This fluorescence polarization immunoassay satisfied the standard criteria of accuracy and precision. The values correlated well with those obtained by radioimmunoassay.     

Differential polarization imaging. I. Theory

A theory of differential polarization imaging is derived using Mueller calculus. It is shown that, for any arbitrary object, 16 images (in general different) can be obtained by combining different incident polarizations of light and measuring the specific polarization components transmitted or scattered by the object. These are called the Mueller images of the object. Mathematical expressions of these images for an object of arbitrary geometry are derived using classical vector diffraction theory and the paraxial and thin lens approximations. The object is described as a collection of point polarizable groups. The electromagnetic fields are calculated using the first Born-Approximation, but extension of the theory to higher-order approximations is shown to be straightforward. These expressions are obtained for the transmission, or bright-field, geometry, and the scattering, or dark-field, configuration. In both cases, the contributions of scattering, absorption, and background illumination to the Mueller images are characterized. The contributions of linear dichroism, circular dichroism, and linear and circular intensity differential scattering to certain Mueller images are established. It is shown that the Mueller images represent a complete two-dimensional mapping of the molecular anisotropy of the object.     

Assay of proteolytic enzymes by the fluorescence polarization technique.

Neuraminidase substrates of high specific activity (>300 μCi/μmol) were prepared by reduction of sialyllactose with NaB³H₄, followed by separation of the 2 → 3 and 2 → 6 isomers of [³H]sialyllactitol by paper chromatography. Hydrolysis of sialyllactitol by neuraminidase was monitored by measuring the radioactivity in the neutral reaction product, which was separated from the charged substrate by passage over a small anion exchange column. The assay was applied to the neuraminidase activity of cultured human skin fibroblasts. The K_m was found to be 1.1 mm for both substrates; the pH optimum, 4.0; the 2 → 3 isomer was hydrolyzed twice as fast as the 2 → 6. In several genetic disorders associated with neuraminidase deficiency, the activity toward both isomers was reduced almost completely (mucolipidoses I and II; Goldberg syndrome), or only partially (mucolipidosis III; adult myoclonus syndrome); however, the relative activity towards the two isomers remained approximately the same in all cases.     

Transient polarization currents in the squid giant axon.

It has been repeatedly noted that the change of conformation of the molecules that serve as the ion-selective channels for sodium and potassium conductance in the nerve membrane will be accompanied by a change in the dipole moment of the molecule. This time-dependent change of dipole moment will produce transient currents in the membrane. The canonical form for these currents is determined with conventional statistical mechanics formalism. It is pointed out that the voltage dependence of the conductance channel conductance determines the free energy of the system to within a factor that is an unknown function of the voltage. Since the dipole currents do not depend on this unknown function, they are completely determined 0y the observed properties of the conductance system. The predicted properties of these dipole currents, their time constants and strengths, are calculated. By using the observed properties of gating currents, the density of the sodium channels is computed. The predicted properties of the dipole currents are found to compare satisfactorily with the observed properties of gating currents.     

Evidence of inherent spontaneous polarization in the metazoan integument epithelia.

The live integument epithelia of the metazoa have an inherent spontaneous polarization (an inherent permanent electric dipole moment) of corresponding direction perpendicular to the integument surface. The existence of the inherent polarization was proved by their temperature dependence, i.e., by the pyroelectric (PE) effect. Quantitative PE measurements were carried out on a number of integument epithelia of vertebrates (a) in vivo, (b) on fresh epidermis preparations, and (c) on dead, air-dried epidermis specimens of the same species. The demonstrated spontaneous polarization is not dependent on the living state and not caused by a potential difference between the outer and inner integument surface. Dead, dry epidermis samples (potential difference less than 0.01 mV) as well as dead, dry integument appendages (bristles, hairs), and dead cuticles (of arthropoda, annelida, nematoda) showed an inherent dipole moment of the same orientation as the live epidermis. The findings reveal a relationship between the direction (vector) of inherent spontaneous polarization and that of growth (morphogenesis) in the animal epidermis, their appendages, and cuticles. We conclude (a) that the inherent spontaneous polarization is present in live individual epithelial cells of the metazoan integument, and (b) that this physical property is related to the structural and functional cell polarity of integument epithelia and possibly of other epithelia.     

Surfing the actomyosin wave: polarization of the C. elegans zygote

Many cell types rely on asymmetrically localized PAR proteins to become polarized. New evidence reveals that cortical flows powered by actomyosin contractions can mobilize PAR complexes to create distinct cortical domains.     

Quantification of protein-protein interactions using fluorescence polarization.

Quantitative determinations of the dissociation constants of biomolecular interactions, in particular protein-protein interactions, are essential for a detailed understanding of the molecular basis of their specificities. Fluorescence spectroscopy is particularly well suited for such studies. This article highlights the theoretical and practical aspects of fluorescence polarization and its application to the study of protein-protein interactions. Consideration is given to the nature of the different types of fluorescence probes available and the probe characteristics appropriate for the system under investigation. Several examples from the literature are discussed that illustrate different practical aspects of the technique applied to diverse systems.     

Wnt-dependent spindle polarization in the early C. elegans embryo

Correct orientation of the mitotic spindle is crucial for the proper segregation of localized determinants and the correct spatial organization of cells in early embryos. The cues dividing cells use to orient their mitotic spindles are currently the subject of intensive investigation in a number of model systems. One of the cues that cells use during spindle orientation is provided by components of the Wnt signaling pathway. Because of its stereotypical cleavage divisions, the availability of Wnt pathway mutants and the ability to perform RNAi, and because cell-cell interactions can be studied in vitro, the C. elegans embryo continues to be a useful system for identifying specific cell-cell interactions in which Wnt-dependent signals polarize the mitotic spindle. This review discusses the evidence for involvement of Wnt signaling during spindle orientation in several contexts in the early C. elegans embryo, a process that involves upstream Wnt effectors but does not involve downstream nuclear effectors of Wnt signaling, and places this Wnt spindle orientation pathway in the larger context of other known modulators of spindle orientation in animal embryos.     

Effects of double-layer polarization on ion transport.

It has been proposed that changes in ionic strength will alter the shape of current-voltage relations for ion transport across a lipid membrane. To investigate this effect, we measured currents across glyceryl monooleate membranes at applied potentials between 10 and 300 mV using either gramicidin and 1 mM NaCl or valinomycin and 1 mM KCl. A bridge circuit with an integrator as null detector was used to separate the capacitative and ionic components of the current. The changes in the current-voltage relations when ionic strength is varied between 1 and 100 mM are compared with predictions of Gouy-Chapman theory for the effects of these variations on polarization of the electrical diffuse double-layer. Double-layer polarization accounts adequately for the changes observed using membranes made permeable by either gramicidin or valinomycin.     

Fluorescence polarization: analysis of carbohydrate-protein interaction.

Fluorescence polarization has been widely used for the studies on the molecular motion in solution and has been applied to immunoassays for proteins, therapeutic drug monitoring in clinical pharmacy, and assays for environmentally toxic compounds. Because fluorescence polarization is most readily applicable to the kinetic analysis of the binding reaction between a substance having small molecular mass and a receptor molecule, this method is easily applied to the analysis of carbohydrate-lectin binding. In this tutorial Thematic Review, we briefly introduce the principles of fluorescence polarization and some applications of fluorescence polarization technique to glycobiology.     

Perceiving polarization with the naked eye: characterization of human polarization sensitivity

Like many animals, humans are sensitive to the polarization of light. We can detect the angle of polarization using an entoptic phenomenon called Haidinger''s brushes, which is mediated by dichroic carotenoids in the macula lutea. While previous studies have characterized the spectral sensitivity of Haidinger''s brushes, other aspects remain unexplored. We developed a novel methodology for presenting gratings in polarization-only contrast at varying degrees of polarization in order to measure the lower limits of human polarized light detection. Participants were, on average, able to perform the task down to a threshold of 56%, with some able to go as low as 23%. This makes humans the most sensitive vertebrate tested to date. Additionally, we quantified a nonlinear relationship between presented and perceived polarization angle when an observer is presented with a rotatable polarized light field. This result confirms a previous theoretical prediction of how uniaxial corneal birefringence impacts the perception of Haidinger''s brushes. The rotational dynamics of Haidinger''s brushes were then used to calculate corneal retardance. We suggest that psychophysical experiments, based upon the perception of polarized light, are amenable to the production of affordable technologies for self-assessment and longitudinal monitoring of visual dysfunctions such as age-related macular degeneration.     

S.E.M. and polarization microscopy of nemertean stylets

The ultrastructure of the stylets produced by nine species of nemerteans has been examined by scanning electron microscopy (S.E.M.) and polarized light microscopy. Stylets are solid, nail-shaped structures that typically reach lengths of 50–200 μm. Each stylet is composed of a centrally located organic matrix surrounded by an inorganic cortex that contains calcium and phosphorus. When viewed at high magnifications, fine granules can be seen throughout the organic matrix, and the cortex appears to be composed of densely packed homo-geneous material. Fractured specimens and whole matrices isolated from decalcified stylets reveal a close correspondence between the shape of the organic matrix and that of the surrounding cortex. This similarity in morphology suggests that the organic matrix serves as a template during calcification of the stylet. The fact that abundant material can be seen in the core of incinerated stylets, and in the central region of stylets that had been soaked for several hours in sodium hypochlorite, supports the hypothesis that the organic matrix is also highly calcified. Polarization microscopy of nemertean stylets indicates that they are composed of a crystalline, rather than amorphous, form of calcium phosphate. The probable organization of the calcium phosphate crystals is discussed.     

Polarization of the intrinsic fluorescence of proteins. I. Factors determining the form of the polarization spectrum

The dependence of the slopes of normalized Perrin plots on the excitation wavelength was established. It was shown that the cause of this effect is the anisotropy of the Brownian rotation of proteins, which must be regarded as asymmetrical particles with specific, but not random, orientation of tryptophan with respect to the main axes of the macromolecule. These findings were analysed on the basis of the rotational depolarization theory of such systems, applied for the case when bands of two absorption oscillators overlap as it is for oscillators 1La and 1Lb in the longest wavelength absorption band of tryptophan. It was shown that anisotropy of Brownian molecular rotation is one of the factors that determines the form of the polarization spectrum. The difference of the polarization spectrum of proteins from that of tryptophan, extrapolated to the infinite viscosity, is determined by energy transfer processes in proteins.     

A theory of fluorescence polarization decay in membranes.

Decay of fluorescence polarization after an impulsive excitation is correlated with wobbling motion of fluorescent molecules in membranes. The motion is characterized by two parameters, a "wobbling diffusion constant" and a "degree of orientational constraint" both of which can be determined directly from experimentally obtained decay. Detailed discussion, including theoretically calculated time-courses of polarization decay, is given for several types of molecules embedded in lipid bilayers; these types cover a large part of fluorescent probes available at present. The theory is useful for the analysis of fluorescence polarization decay in any system where the orientation of fluorophore is restricted by the surrounding structure.     

Differential polarization imaging. II. Symmetry properties and calculations

Various differential polarization images or Mueller images of model objects are generated using the equations derived in the previous paper (paper I of this series). These calculated images include models of the higher-order organization of metaphase chromosomes, and show the applicability of the differential polarization imaging method to the elucidation of complex molecular organizations. Then, the symmetry behavior of the Mueller matrix elements upon infinitesimal rotations of the optical components about the optical axis of the imaging system is presented. It is shown that the rotational properties of the Mueller images can be used to eliminate the linear polarization contributions to the M14 and M44 images, which appear when these images are generated with imperfect circular polarizations. The relationships between the 16 bright-field Mueller images for four different media, i.e., linearly and circularly isotropic, circularly anisotropic, linearly anisotropic, and linearly and circularly anisotropic, are also derived. For the first three cases simple relationships between the Mueller images are found and phenomenological equations in terms of the optical coefficients are derived. In the last case there are no specific relationships between the Mueller images and instead we briefly present Schellman and Jensen's method for treating this type of medium. The criterion of spatial resolution between adjacent domains of different optical anisotropy is then derived. It is found that in transitions between domains of opposite anisotropy the classical Rayleigh limit must be replaced by a magnitude criterion which depends on the limits of the sensitivity of the detection. Finally, the feasibility of optical sectioning in differential polarization imaging is demonstrated.