Fluorescence polarization studies of squid giant axons stained with N-methylanilinonaphthalenesulfonates.

The polarized components of the extrinsic fluorescence of squid giant axons stained with 2,6-MANS or 1,8-MANS were studied. The polarization properties of the fluorescence changes associated with voltage-clamp pulses were found to be very different from those of the static fluorescence, supporting the notion that the optical changes involve highly oriented membrane adsorbed fluorophores. The theoretical expectations according to this hypothesis are discussed in detail. The experimental results are in good agreement with the theory assuming that possible probes reorientations are soley due to the action of the applied electric field upon the probes electric dipole. The quantitative analysis of the data for 2,6-MANS provides a fairly accurate determination of the orientation of the membrane bound 2,6-MANS molecules responsible for the fluorescence changes. Such orientation appears to be independent of the membrane face exposed to staining. The data for 1,8-MANS indicate a very different orientation of this isomer. The results suggest a profitable use of extrinsic fluorophores for studies of the structural organization of nerve membranes.     

Quantitative study of polymer conformation and dynamics by single-particle tracking

We present a new method for analyzing the dynamics of conformational fluctuations of individual flexible polymer molecules. In single-particle tracking (SPT), one end of the polymer molecule is tethered to an immobile substratum. A microsphere attached to the other end serves as an optical marker. The conformational fluctuations of the polymer molecule can be measured by optical microscopy via the motion of the microsphere. The bead-and-spring theory for polymer dynamics is further developed to account for the microsphere, and together the measurement and the theory yield quantitative information about molecular conformations and dynamics under nonperturbing conditions. Applying the method to measurements carried out on DNA molecules provides information complementary to recent studies of single DNA molecules under extensional force. Combining high precision measurements with the theoretical analysis presented here creates a powerful tool for studying conformational dynamics of biological and synthetic macromolecules at the single-molecule level.     

Flow dichroism of capsid DNA phages. I. Fast and slow T4B

Flow linear dichroic studies have been conducted on phage T4B in its fast and slow forms. The behavior of the phages is well represented by an equivalent ellipsoid model using the Peterlin-Stuart theory. The measurements permit the evaluation of the optical factor of the DNA in the phage and the rotary diffusion coefficient of the phage particle. Both these quantities change during the slow–fast conversion. The rotary diffusion results are in good agreement with those obtained by other workers with other methods. The optical factor is negative, indicating a net alignment of DNA helices parallel to the phage axis. The results exclude certain simplified models for the packaged DNA but do not lead to a unique structural conclusion. The flow dichroism experiment and its interpretation are described, and a simple method of calculating optical factors for complicated but cylindrically symmetric structures is presented.     

A simple theory of motor protein kinetics and energetics

A simple stochastic theory for kinetics and energetics of the movement of single motor proteins is presented. The model combines the biochemical cycle of nucleotide hydrolysis with the motor protein translocation. Based on the theory of Markov processes, the model provides the force-velocity relationship, the isometric force, and the stochastic stepping of the motor protein along its one-dimensional track. The theoretical model provides a conceptual framework for realistic studies of motor proteins. Relationship between the present theory and other existing models is discussed.     

Fluorescence polarization studies of squid giant axons stained with N-methylanilinonaphthalenesulfonates

The polarized components of the extrinsic fluorescence of squid giant axons stained with 2,6-MANS or 1,8-MANS were studied. The polarization properties of the fluorescence changes associated with voltage-clamp pulses were found to be very different from those of the static fluorescence, supporting the notion that the optical changes involve highly oriented membrane adsorbed fluorophores. The theoretical expectations according to this hypothesis are discussed in detail. The experimental results are in good agreement with the theory assuming that possible probes reorientations are solely due to the action of the applied electric field upon the probes electric dipole. The quantitative analysis of the data for 2,6 MANS provides a fairly accurate determination of the orientation of the membrane bound 2,6-MANS molecules responsible for the fluorescence changes. Such orientation appears to be independent of the membrane face exposed to staining. The data for 1,8-MANS indicate a very different orientation of this isomer. The results suggest a profitable use of extrinsic fluorophores for studies of the structural organization of nerve membranes.     

Birefringence of tropomyosin crystals.

The birefringence of tropomyosin crystals was measured in the temperature range 5 degrees-35 degrees C. The experimental results are compared with a simple model calculation based on the theory developed by Wiener for the optical properties of colloidal systems. The difference between experimental and theoretical values is less than 15%, which denotes a good agreement given the simplicity of the model. A value of 0.011 was obtained for the intrinsic birefringence of the tropomyosin molecule. The temperature dependence of the crystal birefringence could be accounted for in part by a change of the unit cell parameters; this change was experimentally observed by others in x-ray diffraction experiments.     

Competition between solution and cell surface receptors for ligand. Dissociation of hapten bound to surface antibody in the presence of solution antibody.

We present a joint theoretical and experimental study on the effects of competition for ligand between receptors in solution and receptors on cell surfaces. We focus on the following experiment. After ligand and cell surface receptors equilibrate, solution receptors are introduced, and the dissociation of surface bound ligand is monitored. We derive theoretical expressions for the dissociation rate and compare with experiment. In a standard dissociation experiment (no solution receptors present) dissociation may be slowed by rebinding, i.e., at high receptor densities a ligand that dissociates from one receptor may rebind to other receptors before separating from the cell. Our theory predicts that rebinding will be prevented when S much greater than N2Kon/(16 pi 2D a4), where S is the free receptor site concentration in solution, N the number of free surface receptor sites per cell, Kon the forward rate constant for ligand-receptor binding in solution, D the diffusion coefficient of the ligand, and a the cell radius. The predicted concentration of solution receptors needed to prevent rebinding is proportional to the square of the cell surface receptor density. The experimental system used in these studies consists of a monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), that reversibly binds to a monoclonal anti-DNP immunoglobulin E (IgE). This IgE is both a solution receptor and, when anchored to its high affinity Fc epsilon receptor on rat basophilic leukemia (RBL) cells, a surface receptor. For RBL cells with 6 x 10(5) binding sites per cell, our theory predicts that to prevent DCT rebinding to cell surface IgE during dissociation requires S much greater than 2,400 nM. We show that for S = 200-1,700 nM, the dissociation rate of DCT from surface IgE is substantially slower than from solution IgE where no rebinding occurs. Other predictions are also tested and shown to be consistent with experiment.     

Efficiency of Resonance Energy Transfer in Homo-Oligomeric Complexes of Proteins

A theoretical model is proposed for the apparent efficiency of fluorescence (Förster) resonance energy transfer (FRET) in mixtures of free monomers and homo-oligomeric protein complexes of uniform size. The model takes into account possible pathways for transfer of optical excitations from single donors to multiple acceptors and from multiple donors (non-simultaneously) to single acceptors. This necessary departure from the standard theory has been suggested in the literature, but it has only been successfully implemented for a few particular cases, such as for particular geometries of the oligomers. The predictions of the present theoretical model differ significantly from those of the standard theory, with the exception of the case of dimers, for which agreement is observed. This model therefore provides new insights into the FRET behavior of oligomers comprising more than two monomers, and also suggests means for determining the size of oligomeric protein complexes as well as the proportion of associated and unassociated monomers.     

Real-time fluorescence detection of multiple microscale cell culture analog devices in situ.

We investigated multiple microscale cell culture analog (microCCA) assays in situ with a high-throughput imaging system that provides quantitative, nondestructive, and real-time data on cell viability. Since samples do not move between measurements, captured images allow accurate time-course measurements of cell population response and tracking the fate of each cell type on a quantitative basis. The optical system was evaluated by measuring the short-term response to ethanol exposure and long-term growth of drug-resistant tumor cell lines with simultaneous samples. The optical system based on epi-fluorescent excitation consists of an LED and a CCD as well as discrete optical components for imaging a large number of cells simultaneously. HepG2/C3A and MESSA cell lines were cultured in two microCCA systems for continuous cell status monitoring in cell death experiments with ethanol and long-term cell growth. The experiment that tested ethanol uptake showed that ethanol immediately caused cell death. The system was applied to extracting dynamic constants in the uptake process. In the long-term cell growth experiment, growth of MESSA cells was followed by a stationary phase and eventual cell death attributed to nutrient and oxygen depletion and a change in the pH because of the accumulation of wastes by cell metabolism. HepG2/C3A cells were subject to contact inhibition and cell number did not change significantly over time. Issues related to long-term assays are also discussed. The quantitative results have been consistent with qualitative images and confirm the applicability of the portable optical system, and potential application to high-throughput analysis of cell-based assays to measure long-term dynamics.     

Bird flight and optimal migration

The development of the mechanical and aerodynamical theory of bird flight has greatly stimulated research at widely different levels in the field of bird movement. Recent work has shown that the drag of bird bodies is less than was previously assumed. Furthermore, the structure and circulation of wingtip vortices in the wake of flying birds have been revealed, with implications for estimating flight performance on the basis of vortex theory. Predictions about optimal speed and flight behaviour have been successfully tested by field studies using optical and radar registration. Flight theory also allows predictions about optimal fuel deposition rules for migrating birds. Research about bird flight, with the dynamic interplay between theoretical development and empirical work in biophysics, physiology and ecology, represents a fine example of a highly successful use of the optimality approach in biology.     

Probability density function learning by unsupervised neurons.

In a recent work, we introduced the concept of pseudo-polynomial adaptive activation function neuron (FAN) and presented an unsupervised information-theoretic learning theory for such structure. The learning model is based on entropy optimization and provides a way of learning probability distributions from incomplete data. The aim of the present paper is to illustrate some theoretical features of the FAN neuron, to extend its learning theory to asymmetrical density function approximation, and to provide an analytical and numerical comparison with other known density function estimation methods, with special emphasis to the universal approximation ability. The paper also provides a survey of PDF learning from incomplete data, as well as results of several experiments performed on real-world problems and signals.     

Transmembrane Protein Diffusion in Gel-Supported Dual-Leaflet Membranes

Tools to measure transmembrane-protein diffusion in lipid bilayer membranes have advanced in recent decades, providing a need for predictive theoretical models that account for interleaflet leaflet friction on tracer mobility. Here we address the fully three-dimensional flows driven by a (nonprotruding) transmembrane protein embedded in a dual-leaflet membrane that is supported above and below by soft porous supports (e.g., hydrogel or extracellular matrix), each of which has a prescribed permeability and solvent viscosity. For asymmetric configurations, i.e., supports with contrasting permeability, as realized for cells in contact with hydrogel scaffolds or culture media, the diffusion coefficient can reflect interleaflet friction. Reasonable approximations, for sufficiently large tracers on low-permeability supports, are furnished by a recent phenomenological theory from the literature. Interpreting literature data, albeit for hard-supported membranes, provides a theoretical basis for the phenomenological Stokes drag law as well as strengthening assertions that nonhydrodynamic interactions are important in supported bilayer systems, possibly leading to overestimates of the membrane/leaflet viscosity. Our theory provides a theoretical foundation for future experimental studies of tracer diffusion in gel-supported membranes.     

Probing field-induced tissue polarization using transillumination fluorescent imaging

Despite major successes of biophysical theories in predicting the effects of electrical shocks within the heart, recent optical mapping studies have revealed two major discrepancies between theory and experiment: 1), the presence of negative bulk polarization recorded during strong shocks; and 2), the unexpectedly small surface polarization under shock electrodes. There is little consensus as to whether these differences result from deficiencies of experimental techniques, artifacts of tissue damage, or deficiencies of existing theories. Here, we take advantage of recently developed near-infrared voltage-sensitive dyes and transillumination optical imaging to perform, for the first time that we know of, noninvasive probing of field effects deep inside the intact ventricular wall. This technique removes some of the limitations encountered in previous experimental studies. We explicitly demonstrate that deep inside intact myocardial tissue preparations, strong electrical shocks do produce considerable negative bulk polarization previously inferred from surface recordings. We also demonstrate that near-threshold diastolic field stimulation produces activation of deep myocardial layers 2-6 mm away from the cathodal surface, contrary to theory. Using bidomain simulations we explore factors that may improve the agreement between theory and experiment. We show that the inclusion of negative asymmetric current can qualitatively explain negative bulk polarization in a discontinuous bidomain model.     

Use of implicit methods from general sensitivity theory to develop a systematic approach to metabolic control. I. Unbranched pathways

It is shown that metabolic control theory (MCT), is its present form, is a particular case of general sensitivity theory, which studies the effects of parameter variations on the behavior of dynamic systems. It has been shown that metabolic control theory is obtained from this more general theory for the particular case of steady-state and linear relationships between velocities and enzyme concentrations. In such conditions the relationships between elasticities and flux control coefficients are easily obtained. These relationships are in the form of a matrix product constructed in a priori form. Relationships between combined response coefficients and concentration control coefficients are presented. The use of implicit methodology from general sensitivity theory provides a generalization of MCT, which is applied to unbranched pathways. For this particular case, provided the matrices have been properly constructed, the matrix of global properties (flux and concentration control coefficients) can be obtained by inversion of the matrix of local properties (elasticities). The theorems of MCT (concentration summation, flux summation, flux connectivity, and concentration connectivity) applicable for unbranched pathways are directly obtained by inspection of the matrix product. With these results, the present theoretical basis of MCT is extended with a more structured framework that allows a wider range of application. The results make clearer the relatedness of MCT to the more general approach provided by biochemical systems theory (BST).     

SPR-based DNA Detection with Metal Nanoparticles

In this short review paper, we summarize some of our ideas to utilize gold nanoparticles for the enhancement of surface plasmon resonance signals on DNA microarray. The hybridization of target-DNA capped gold nanoparticles with probe DNA on surface provides ca. ten times stronger optical contrast compared with that of target-DNA molecules. Our simulation result based on the Maxwell-Garnet theory explains well our experimental data and proves a potential of metallic nanoparticles for the substantial sensitivity enhancements for biosensor application in DNA diagnostics and bio-affinity studies, which leads to the fabrication of high resolution DNA microarrays.     

Density functional theory calculations of optical rotation: employment of ADZP and its comparison with other basis sets

Density function theory calculations of frequency dependent optical rotations ([alpha]omega) for 30 rigid chiral molecules are reported. Calculations have been carried out at the sodium D line frequency, using the augmented double zeta valence quality plus polarization functions (ADZP) basis set and the BP86 nonhybrid and B3LYP hybrid functionals. Gauge-invariant atomic orbitals were used to guarantee origin-independent values of [alpha]D. Comparison between corresponding results obtained with nonhybrid and hybrid functionals as well as with theoretical optical rotations reported in the literature is done. Excited electronic states of three molecules are also discussed in light of circular dichroism spectra and B3LYP and BP86 calculated excitation energies and rotatory strengths. One verifies that the B3LYP/ADZP results are in better agreement with experiment.     

The molecular origin of birefringence in skeletal muscle. Contribution of myosin subfragment S-1.

The state of optical polarization of He-Ne laser light diffracted by single skinned frog skeletal muscle fibers has been determined after decoration of the thin filaments of rigor fibers with exogenous S-1. Light on the first diffraction order was analyzed using optical ellipsometry for changes occurring in total birefringence (delta nT) and total differential field ratio (rT) and the experimental results compared with theoretical predictions. Fibers were examined with SDS-gel electrophoresis and electron microscopy as independent assays of S-1 binding. The binding of S-1 to the thin filaments caused a significant increase in rT and a small but significant decrease in delta nT. Release of bound exogenous S-1 with magnesium pyrophosphate demonstrated that the effect of S-1 on the optical parameters was reversible and both electrophoresis and electron microscopy demonstrated the presence of S-1 specifically bound to the thin filaments. Model simulations based on the theory of Yeh, Y., and R. Baskin (1988. Biophys. J. 54:205-218) showed that the values of delta nT and rT were sensitive to the axial bonding angle of exogenous S-1 as well as to the volume fraction of added S-1. Analysis of the data in light of the model showed that an average axial S-1 binding angle of 68 degrees +/- 7 degrees best fit the data.     

Dielectric analysis of mitochondria isolated from rat liver. I. Swollen mitoplasts as simulated by a single-shell model

A re-evaluation of the dielectric studies on isolated mitochondria (Pauly, H., Packer, L. and Schwan, H.P. (1960) J. Biophys. Biochem. Cytol. 7, 589-601, and ibid. 7, 603-612) is presented. The suspensions of 'mitoplasts' prepared from rat liver mitochondria by a hyposmotic (10 mM KCl) treatment showed a dielectric dispersion with its characteristic frequency lying in the 1-100 MHz range. In the analysis of data special emphasis was put on the choice of the theoretical models to employ after scrutiny of their applicability to the suspensions tested. As such we adopted the theory of Hanai et al. (Hanai, T., Asami, K., and Koizumi, N. (1979) Bull. Inst. Chem. Res., Kyoto Univ. 57, 297-305) that was advanced to include concentrated suspensions of shelled spheres. Curve fittings based on that theory resulted in a better agreement with experiment than the fittings based on a conventional theory for dilute suspensions. Major findings from our analyses on the swollen mitoplasts are that: (i) the limiting membrane of the mitoplasts has a specific electrical capacity of 1 microF/cm2, (ii) the ratio of permittivity (or dielectric constant) for the mitoplast interior and permittivity for the external medium is 0.6-0.7, and (iii) the conductivity ratio between the interior phase and the medium is approx. 0.6. Reasons for discrepancy between the results of Pauly et al. and ours are discussed.     

Tunable Plasmon–Induced Transparency Based on Dirac Semimetals

Numerical and theoretical studies were conducted on the plasmon induced transparency (PIT) of the symmetrical structure of Dirac semi-metal films (DSFS). The films have a parallel strip and split resonant ring structure. After analysing the surface current intensity and distribution, it was found that the electromagnetically induced transparency is as a result of destructive interference between these two structures, with the amplitude modulation depth of the frequency of the transmission window reaching as high as 99.09%. Moreover, by adjusting the Fermi level of the DSFS, the Fermi level changed from 50 to 90 meV, and the transmission window blue-shifted from 0.529 to 0.799 THz. The transmission peak frequency was found to have a linear relationship with the Fermi level. Similarly, the transmission phase and group delay under different Fermi levels was investigated. The positive group delay of the film reaches 7.026 ps, which provides a direction for new applications of terahertz, such as optical storage and slow optical devices.

     

Solution structure and dynamics of biomolecules from Raman optical activity

Raman optical activity (ROA) measures vibrational optical activity by means of a small difference in the intensity of Raman scattering from chiral molecules in right and left circularly polarized incident laser light. The ROA spectra of a wide range of biomolecules in aqueous solution can now be measured routinely. Because of its sensitivity to the chiral elements of biomolecular structure, ROA provides new information about solution structure and dynamics complementary to that supplied by conventional spectroscopic techniques. This article provides a brief introduction to the theory and practice of ROA spectroscopy followed by a review of recent ROA results on polypeptides, proteins, carbohydrates, nucleic acids and viruses which illustrate how new insight into current problems of structure, folding and function may be obtained from ROA studies.