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B A Scalettar

Publications and source records attributed to B A Scalettar.

10 recordsLinked to original sources
PubMed1992

Evanescent interference patterns for fluorescence microscopy.

The increasing experimental use of total internal reflection/fluorescence photobleaching recovery has motivated a theoretical study of the spatial intensity profiles generated by two interfering evanescent waves. The interference patterns generated by evanescent waves differ considerably from those generated by plane waves in a homogenous medium because evanescent waves are not transverse and because the evanescent propagation number depends on the incidence angle of the totally internally reflected light. The periodicity and contrast of the evanescent interference patterns under various conditions are calculated; these parameters depend on the intensities, polarizations, and incidence angles of the two incident beams, as well as the refractive indices of the two media that form the planar interface where total internal reflection occurs. The derived intensity profiles are used to develop expressions for the shapes of fluorescence photobleaching recovery curves when evanescent interference patterns are used for fluorescence excitation and bleaching. The calculations also suggest that colliding beam experiments may confirm theoretically predicted evanescent field polarizations.

Binding Sites↗
PubMed1991

Dynamics, structure, and function are coupled in the mitochondrial matrix.

The coupling between molecular diffusion and the structure and function of the rat liver mitochondrial matrix was explored using fluorescence anisotropy techniques and electron microscopy. The results confirm that matrix ultrastructure and the concentration of matrix protein are influenced by the respiratory state of mitochondria and the osmolarity of the external medium. At physiological osmolarity, a fluorescent metabolite-sized probe was found to diffuse slowly in the mitochondrial matrix but not to be completely immobile. In addition, significant differences in diffusion rates were found to exist between different mitochondrial respiratory states, with the slowest diffusion occurring in states with the highest matrix protein concentration. These data support the concept of a matrix structure in which diffusion is considerably hindered due to limited probe-accessible water and further suggest that volume-dependent regulation of matrix protein packing may modulate metabolite diffusion and, in turn, mitochondrial metabolism.

Animals↗
PubMed1990

A polarized photobleaching study of chromatin reorientation in intact nuclei.

Polarized fluorescence recovery after photobleaching (pFRAP) was used to monitor the effects that condensation, i.e. compaction and aggregation, have on the (microseconds and ms) internal dynamics of chromatin in intact nuclei. When divalent cations were present with physiological (approximately 90 mM) monovalent salt the chromatin was found to exist in a compact and aggregated state which was characterized by rotational immobilization over timescales that range from 10 microseconds to 40 milliseconds. This immobilization is attributed to suppression of internal dynamics by intermolecular interactions. When the divalent cations were removed, the compact fibers no longer aggregated and were free to reorient with a characteristic decay time of about 1.2 milliseconds. It is shown that this millisecond relaxation could represent rigid rotation of topologically independent structural domains. Dilution of the monovalent salt induced a gradual change in the structural state of the chromatin that was manifest as a dramatic increase in internal flexibility. At the lowest salt concentration studied (11 mM-monovalent salt) the chromatin reorients in fewer than ten microseconds. These changes in flexibility are continuous with salt concentration, indicating that there are no well-defined endpoints to structural transitions and that the microsecond-millisecond internal dynamics of chromatin are a sensitive measure of structure. Measurements made on nuclei from cells that are either transcriptionally quiescent or active indicate that the dynamics mirrors biological activity.

Animals↗
PubMed1990

A polarized photobleaching study of DNA reorientation in agarose gels.

Polarized fluorescence recovery after photobleaching (pFRAP) has been used to study the internal dynamics of relatively long DNA molecules embedded in gels that range in concentration from 1% to 5% agarose. The data indicate that, even in very congested gels, rapid internal relaxation of DNA is largely unhindered; however, interactions with gel matrices apparently do perturb the larger amplitude, more slowly (microseconds to milliseconds) relaxing internal motions of large DNAs. The relationship between this work and recent studies which indicate that internal motions of DNA play an important role in the separation achieved with pulsed-field gel electrophoresis techniques is discussed. The polarized photobleaching technique is also analyzed in some detail. In particular, it is shown that "reversible" photobleaching phenomena are probably related to depletion of the ground state by intersystem crossing to the triplet state.

DNA↗
PubMed1990

On the measurement of particle number and mobility in nonideal solutions by fluorescence correlation spectroscopy.

Interparticle interactions are incorporated into the theoretical description of the initial amplitude, G(0), of the normalized fluorescence correlation spectroscopy autocorrelation function. Measurements of particle number, aggregate size, and interaction-dependent diffusion are then analyzed in the context of this generalized theory. It is shown that the neglect of interactions can introduce order-of-magnitude errors into estimates of particle number and aggregate size. It is also shown that measurement of G(0) provides an essentially unique method for testing the validity of theories of interaction-dependent membrane protein diffusion.

Mathematics↗
PubMed1989

Mutual diffusion of interacting membrane proteins.

The generalized Stokes-Einstein equation is used, together with the two-dimensional pressure equation, to analyze mutual diffusion in concentrated membrane systems. These equations can be used to investigate the role that both direct and hydrodynamic interactions play in determining diffusive behavior. Here only direct interactions are explicitly incorporated into the theory at high densities; however, both direct and hydrodynamic interactions are analyzed for some dilute solutions. We look at diffusion in the presence of weak attractions, soft repulsions, and hard-core repulsions. It is found that, at low densities, attractions retard mutual diffusion while repulsions enhance it. Mechanistically, attractions tend to tether particles together and oppose the dissipation of gradients or fluctuations in concentration, while repulsions provide a driving force that pushes particles apart. At higher concentrations, changes in the structure of the fluid enhance mutual diffusion even in the presence of attractions. It is shown that the theoretical description of postelectrophoresis relaxation and fluorescence correlation spectroscopy experiments must be modified if interacting systems are studied. The effects of interactions on mutual diffusion coefficients have probably already been seen in postelectrophoresis relaxation experiments.

Diffusion↗
PubMed1989

Self diffusion of interacting membrane proteins.

A two-dimensional version of the generalized Smoluchowski equation is used to analyze the time (or distance) dependent self diffusion of interacting membrane proteins in concentrated membrane systems. This equation provides a well established starting point for descriptions of the diffusion of particles that interact through both direct and hydrodynamic forces; in this initial work only the effects of direct interactions are explicitly considered. Data describing diffusion in the presence of hard-core repulsions, soft repulsions, and soft repulsions with weak attractions are presented. The effect that interactions have on the self-diffusion coefficient of a real protein molecule from mouse liver gap junctions is also calculated. The results indicate that self diffusion is always inhibited by direct interactions; this observation is interpreted in terms of the caging that will exist at finite protein concentration. It is also noted that, over small distance scales, the diffusion coefficient is determined entirely by the very strong Brownian forces; therefore, as a function of displacement the self-diffusion coefficient decays (rapidly) from its value at infinite dilution to its steady-state interaction-averaged value. The steady-state self-diffusion coefficient describes motion over distance scales that range from approximately 10 nm to cellular dimensions and is the quantity measured in fluorescence recovery after photobleaching experiments. The short-ranged behavior of the diffusion coefficient is important on the interparticle-distance scale and may therefore influence the rate at which nearest-neighbor collisional processes take place. The hard-disk theoretical results presented here are in excellent agreement with lattice Monte-Carlo results obtained by other workers. The concentration dependence of experimentally measured diffusion coefficients of antibody-hapten complexes bound to the membrane surface is consistent with that predicted by the theory. The variation in experimental diffusion coefficients of integral membrane proteins is greater than that predicted by the theory, and may also reflect protein-induced perturbations in membrane viscosity.

Animals↗
PubMed1988

A fluorescence photobleaching study of the microsecond reorientational motions of DNA.

We have conducted a polarized fluorescence photobleaching recovery (FPR) study of the rotational dynamics of ethidium azide labeled DNA. Polarized photobleaching experiments provide data on microsecond and millisecond molecular reorientation that complement the information available from nanosecond fluorescence depolarization studies. In polarized FPR experiments an anisotropic angular concentration of fluorophore is created by bleaching dye molecules in a preferred orientation with a short, intense pulse of polarized light. The sample is then weakly illuminated, and the temporal variation in the emitted fluorescence is monitored. The fluorescence signal will systematically change as molecules undergo post-bleach reorientation and the angular distribution of dye tends toward isotropy. We have observed that the time dependence of our microsecond FPR curves is also determined in part by nonrotational phenomena. To isolate the reorientational recovery we conduct our FPR experiments in two modes (called parallel and perpendicular) that differ only in the polarization of the bleaching light. A quotient function, R(t), is constructed from the data obtained in these two modes; the variation with time of this new quantity is governed solely by processes that are sensitive to the polarization of the incident light (e.g., molecular rotation). It is found experimentally that R(t) remains constant, as expected, for rotationally restricted DNA systems despite a temporal recovery in the parallel and perpendicular FPR curves. We also follow the dynamics of solutions of phage lambda DNA as revealed in the temporal dependence of R(t). This DNA system rotationally relaxes after approximately 100 microseconds and the dye/DNA complex reorients substantially during the 10-microseconds bleach period. Our FPR data are interpreted in terms of dynamic models of DNA motion.

Azides↗
PubMed1988

Theoretical comparison of the self diffusion and mutual diffusion of interacting membrane proteins.

Self diffusion and mutual diffusion in two-dimensional membrane systems are analyzed. It is shown that interprotein interactions can produce markedly different density-dependent changes in the diffusion coefficients describing these two processes; the qualitative differences are illustrated by using a theoretical formalism valid for dilute solutions. Results are obtained for three analytical potentials: hard-core repulsions, soft repulsions, and soft repulsions with weak attractions. Self diffusion is inhibited by all three interactions. In contrast, mutual diffusion is inhibited by attractions but is enhanced by repulsions. It is shown that such interaction-dependent differences in self diffusion and mutual diffusion could underlie, among other things, the disparity in protein diffusion coefficients extracted from fluorescence recovery after photobleaching and postelectrophoresis relaxation data.

Algorithms↗