Ethambutol-induced alterations in Mycobacterium bovis BCG imaged by atomic force microscopy

Progress in understanding the structure-function relationships of the mycobacterial cell wall has been hampered by its complex architecture as well as by the lack of sensitive, high-resolution probing techniques. For the first time, we used atomic force microscopy (AFM) to image the surface topography of hydrated Mycobacterium bovis bacillus Calmette Guérin cells and to investigate the influence of the antimycobacterial drug ethambutol on the cell wall architecture. While untreated cells showed a very smooth and homogeneous surface morphology, incubation of cells in the presence of ethambutol caused dramatic changes of the fine surface structure. At 4 micro g mL(-1), the drug created concentric striations at the cell surface and disrupted a approximately 8 nm thick cell wall layer, attributed to the outer electron-opaque layer usually seen by electron microscopy, while at 10 micro g mL(-1) an underlying approximately 12 nm thick layer reflecting the thick electron-transparent layer was also altered. These noninvasive ultrastructural investigations provide novel information on the macromolecular architecture of the mycobacterial envelope as well as into the destructuring effects of ethambutol.     

Using continuous porous silicon gradients to study the influence of surface topography on the behaviour of neuroblastoma cells

The effects of surface topography on cell behaviour are the subject of intense research in cell biology. These effects have so far only been studied using substrate surfaces of discretely different topography. In this paper, we present a new approach to characterise cell growth on porous silicon gradients displaying pore sizes from several thousands to a few nanometers. This widely applicable format has the potential to significantly reduce sample numbers and hence analysis time and cost. Our gradient format was applied here to the culture of neuroblastoma cells in order to determine the effects of topography on cell growth parameters. Cell viability, morphology, length and area were characterised by fluorescence and scanning electron microscopy. We observed a dramatic influence of changes in surface topography on the density and morphology of adherent neuroblastoma cells. For example, pore size regimes where cell attachment is strongly discouraged were identified providing cues for the design of low-fouling surfaces. On pore size regimes more conducive to cell attachment, lateral cell-cell interactions crosslinked the cell layer to the substratum surface, while direct substrate-cell interactions were scarce. Finally, our study revealed that cells were sensitive to nanoscale surface topography with feature sizes of <20 nm.     

Atomic force microscopy comes of age

AFM (atomic force microscopy) analysis, both of fixed cells, and live cells in physiological environments, is set to offer a step change in the research of cellular function. With the ability to map cell topography and morphology, provide structural details of surface proteins and their expression patterns and to detect pico‐Newton force interactions, AFM represents an exciting addition to the arsenal of the cell biologist. With the explosion of new applications, and the advent of combined instrumentation such as AFM—confocal systems, the biological application of AFM has come of age. The use of AFM in the area of biomedical research has been proposed for some time, and is one where a significant impact could be made. Fixed cell analysis provides qualitative and quantitative subcellular and surface data capable of revealing new biomarkers in medical pathologies. Image height and contrast, surface roughness, fractal, volume and force analysis provide a platform for the multiparameter analysis of cell and protein functions. Here, we review the current status of AFM in the field and discuss the important contribution AFM is poised to make in the understanding of biological systems.     

Kinetics of a low-density plasma near a dielectric surface with account for secondary electron emission

Exact steady solutions in a one-dimensional kinetic model of the processes in a low-density plasma layer near a dielectric surface are constructed analytically with allowance for secondary electron emission. It is shown that, for low electron temperatures, the solutions describe a regime in which the electric potential and electron density decrease monotonically toward the dielectric wall (a classical Debye layer). For higher electron temperatures, there are solutions describing regimes such that the electric potential and electron density increase monotonically toward the wall (an inverse Debye layer).     

Cell adhesion and cell surface topography in aggregates of 3T3 and SV40-virus-transformed 3T3 cells. Visualization of interior cells by scanning electron microscopy

A technique for exposing the interior of aggregates of cultured cells has been developed and is described in this report. Using this technique, we have examined for the first time, by scanning electron microscopy, cell morphology and cell contact ultrastructure in the interior of aggregates of BALB/c 3T3 and SV40-transformed 3T3 cells. The 3T3 cells make initial intercellular contact by means of microvillar processes. Over a period of 3-8 h, some of these microvillar contacts are replaced by broader projections. In contrast, the SV40-transformed cells make initial intercellular contact by means of blebs or blunt projections which are also broadened and extended over a period of 3-8 h. For both 3T3 and SV40-3T3 cells, the surfaces of the cells which form the outer layer of the aggregate resemble the surfaces of single cells fixed in suspension, regardless of how long the aggregates have been cultured. Thse cells are covered with many cellular processes and are roughly hemispherical in profile. The surfaces of the internal cells of the aggregates, however, lose many of their cellular processes, develop smooth patches, and many become irregular in shape. This smooth morphology was also observed on the interior surfaces of the peripheral cell layer. From these observations we conclude that: (a) the stabilization of adhesive contacts is a slow process which takes at least 3-8 h; (b) the outer surfaces of peripheral cells differ significantly from the surfaces of interior cells; and (c) clear differences in surface topography exist between nonmalignant 3T3 cells and their malignant SV40 transformants.     

Total Hourglass Control - A Robust Finite Element Formulation for the Calculation of Organ Deformations

For use in a virtual reality based laparoscopic surgery simulator methods have been developed to model the mechanical behavior of soft tissue. Explicit Finite Element analysis turned out to be a robust basis when the approximation of hyperelastic materials is used. To reduce calculation times reduced volume integration schemes were applied, where classical hourglass control leads to drastic errors which make a simulation expanding over a long period of time impossible. Therefore, the total hourglass control scheme was developed and is presented here for several material laws. This new formulation takes the existence of the elastic potential of a hyperelastic material into account and leads to robust simulations without loss of accuracy. This is demonstrated with the off-line simulation of a model of an uterus and its adnexe.     

A theoretical model of LDL-receptor trapping on a spherical cell.

The kinetics of the trapping of LDL-receptor complexes by coated pits on the surface of fibroblasts is examined in this paper. We have recently developed a mathematical formalism to extend Keizer's non-linear, non-equilibrium fluctuation-dissipation theory to the kinetics of chemical systems constrained to a spherical surface. Keizer's theory is ideally suited to the study of open biological systems. In the past it has been used to investigate endocytosis on fibroblasts. However, these applications have modeled the cell membrane with an infinite plane. As such, the finite size of the cellular membrane, as well as its precise symmetry, could not be incorporated into the previous studies. Thus in this paper we use our recently developed methodology to reexamine the trapping step in endocytosis on spherical cells. For cell surface processes, the theoretical consideration of a spherical symmetry or an infinite plane, in model calculations, will depend on the experimental or in vivo conditions of the processes of interest. For a spherical symmetry, we find that the finite size of the cell surface does not significantly affect the rate of the trapping step given the empirically determined values for the relevant parametes on fibroblasts. This result supports the approximation used in the previous investigation. However, this and other analyses indicate that the finitie size of the biological surface probably is an important parameter for processes which occur on smaller biological surfaces such as those found on organelles.     

CYCLIC CHANGES IN THE CELL SURFACE : II. The Effect of Cytochalasin B on the Surface Morphology of Synchronized Chinese Hamster Ovary Cells

The surface morphology of Chinese hamster ovary cells treated with cytochalasin B (CB) has been examined using the scanning electron microscope. The cells respond to treatment with CB by retracting peripheral processes, rounding up, and assuming a smooth or gently convoluted surface. This response occurs within minutes. Cells in different stages of the cell cycle all respond in a similar manner. When CB is removed from treated cells by washing with conditioned medium, the cells regain their normal surface conformation within minutes. The surface topography of these released cells is characteristic of their stage in the cell cycle. Because CB causes an alteration in the morphology of the cell surface and because of the speed of the response and recovery, it is proposed that the primary site of action of CB is the cell surface.     

Immobilized particles in gel matrix-type porous media. Nonhomogeneous cell distribution

The conventional random pore model assumes a homogeneous cell distribution in the gel matrix used to immobilize cells. However, the validity of this model is restricted to values of the exponent alpha, between 1.8 and 2.25, of a model power function relating the diffusivity coefficient in the matrix with the overall cell volume fraction in the system. Based on the analysis of published data for diffusion in gels with immobilized cells and on the homogeneous approach for the random pore model developed in a previous work, a new, nonhomogeneous approach is proposed for alpha values outside the range 1.8-2.25. To explain these data, two main types of nonhomogeneous cell distribution were considered: (1) nonhomogeneous cell distribution in the gel for alpha > 2.25 (type 1) and (2) nonhomogeneity related with anisotropy of cell space orientation when alpha < 1.8 (type 2). In the case of nonhomogeneity of type 1, the cell volume fraction in the layers occupied by cells must be considered in place of the concept previously used for homogeneous distribution, viz., the average cell volume fraction. This model underlines that accumulation of cells in a thin layer close to the surface improves their nutrient intake. For nonhomogeneity of type 2, the tortuosity of such a system is smaller than should be expected if spherical cells were considered, thereby changing the effective diffusion. The model proposed in this work proved to fit into several real cases reported in the literature.     

Use of Surface Enhanced Blocking (SEB) Electrodes for Microbial Cell Lysis in Flow-Through Devices

By simultaneously subjecting microbial cells to high amplitude pulsed electric fields and flash heating of the cell suspension fluid, effective release of intracellular contents was achieved. The synergistic effect of the applied electric field and elevated temperature on cell lysis in a flow-through device was demonstrated for Gram-negative and Gram-positive bacteria, and Mycobacterium species. The resulting lysate is suitable for downstream nucleic acid amplification and detection without requiring further preparation. The lysis chamber employs surface enhanced blocking electrodes which possess an etched micro-structured surface and a thin layer of dielectric metal oxide which provides a large effective area and blocks transmission of electrical current. The surface enhanced blocking electrodes enable simultaneous suppression of the rapid onset of electric field screening in the bulk of the cell suspension medium and avoidance of undesired electrochemical processes at the electrode-electrolyte interface. In addition the blocking layer ensures the robustness of the cell lysis device in applications involving prolonged flow-through processing of the microbial cells.     

Topographical control of cell behaviour. I. Simple step cues

The photolithographic techniques of the microelectronics industry have allowed us to fabricate patterned plastic substrata to investigate contact guidance of animal tissue cells. The reactions of cells to single steps on a substratum were examined using time-lapse videorecording and scanning electron microscopy. BHK cells and chick embryonic neural cell processes exhibited gradual inhibition of crossing steps with a concomitant increase in alignment at steps dependent on increasing step height. Comparison of these cells' reactions, with those of chick heart fibroblasts and rabbit neutrophils, at a 5 micron step revealed that the influence of topography is also dependent on cell type, the neutrophils being relatively unaffected. The presence of an adhesive difference at a series of steps altered BHK cells' reactions such that the frequency of crossing was dependent on the direction of approach to a step. Although our data are consistent with Dunn & Heath's proposal (1976) that the inflexibility of the cytoskeleton of a moving cell's protrusion is the cellular property determining such reactions to topography, we have found that, on encountering a topographical feature, the response of a cell may be predictable on a probabilistic basis, i.e. the topographical feature reduces the probability of a cell making a successful protrusion and contact in a given direction, that even the largest features tested did not act as absolute barriers to cell locomotion since a small proportion of a population of cells were able to overcome them, and that other guidance cues could significantly alter a cell's response. Even in situations where it is not the primary cue in directing cell locomotion, topographical control may be an important factor during morphogenesis since it must, at the very least, influence the efficiency of other cues.     

Exact digital simulation of time-invariant linear systems with applications to neuronal modeling

An efficient new method for the exact digital simulation of time-invariant linear systems is presented. Such systems are frequently encountered as models for neuronal systems, or as submodules of such systems. The matrix exponential is used to construct a matrix iteration, which propagates the dynamic state of the system step by step on a regular time grid. A large and general class of dynamic inputs to the system, including trains of δ-pulses, can be incorporated into the exact simulation scheme. An extension of the proposed scheme presents an attractive alternative for the approximate simulation of networks of integrate-and-fire neurons with linear sub-threshold integration and non-linear spike generation. The performance of the proposed method is analyzed in comparison with a number of multi-purpose solvers. In simulations of integrate-and-fire neurons, Exact Integration systematically generates the smallest error with respect to both sub-threshold dynamics and spike timing. For the simulation of systems where precise spike timing is important, this results in a practical advantage in particular at moderate integration step sizes. Received: 3 October 1998 / Accepted in revised form: 19 March 1999     

Role of the S layer in morphogenesis and cell division of the archaebacterium Methanocorpusculum sinense.

Thin sections, freeze-etched, and negatively stained preparations of Methanocorpusculum sinense cells reveal a highly lobed cell structure with a hexagonally arranged surface layer (S layer). Digital image processing of negatively stained envelope fragments show that the S layer forms a porous but strongly interconnected network. Since the S layer is the exclusive cell envelope component outside the cytoplasmic membrane it must have a cell shape determining and maintaining function. Although lattice faults such as disclinations and dislocations are a geometrical necessity on the surface of a closed protein crystal, our data indicate that they also play important roles as sites for the incorporation of new morphological units, in the formation of the lobed cell structure, and in the cell division process. In freeze-etched preparations of intact cells numerous positive and negative 60 degree wedge disclinations can be detected which form pentagons and heptagons in the hexagonal array. Complementary pairs of pentagons and heptagons are the termination points of edge dislocations. They can be expected to function both as sites for incorporation of new morphological units into the lattice and as initiation points for the cell division process. The latter is determined by the ratio between the increase of protoplast volume and the increase in actual S-layer surface area during cell growth. We postulate that this mode of cell fission represents a common feature in lobed archaebacteria which possess an S layer as the exclusive wall component.     

Reduced Model and Simulation of Neuron with Passive Dendritic Cable: An Eigenfunction Expansion Approach

The neuron models with passive dendritic cables are often used for detailed cortical network simulations (Protopapas et al., 1998; Suarez et al., 1995). For this, the compartment model based on finite volume or finite difference discretization was used. In this paper, we propose an eigenfunction expansion approach combined with singular perturbation and demonstrate that the proposed scheme can achieve an order of magnitude accuracy improvement with the same number of equations. Moreover, it is also shown that the proposed scheme converges much faster to attain a given accuracy. Hence, for a network simulation of the neurons with passive dendritic cables, the proposed scheme can be an attractive alternative to the compartment model, that leads to a low order model with much higher accuracy or that converges faster for a given accuracy.     

FINE STRUCTURE AND MORPHOGENIC MOVEMENTS IN THE GASTRULA OF THE TREEFROG, HYLA REGILLA

The blastoporal groove of the early gastrula of the treefrog, Hyla regilla, was examined with the electron microscope. The innermost extension of the groove is lined with invaginating flask- and wedge-shaped cells of entoderm and mesoderm. The distal surfaces of these cells bear microvilli which are underlain with an electron-opaque layer composed of fine granular material and fibrils. The dense layer and masses of vesicles proximal to it fill the necks of the cells. In flask cells bordering the forming archenteron the vesicles are replaced by large vacuoles surrounded by layers of membranes. The cells lining the groove are tightly joined at their distal ends in the region of the dense layer. Proximally, the cell bodies are separated by wide intercellular spaces. The cell body, which is migrating toward the interior of the gastrula, contains the nucleus plus other organalles and inclusions common to amphibian gastrular cells. A dense layer of granular material, vesicles, and membranes lies beneath the surface of the cell body and extends into pseudopodium-like processes and surface undulations which cross the intercellular spaces. A special mesodermal cell observed in the dorsal lining of the groove is smaller and denser than the surrounding presumptive chordamesodermal cells. A long finger of cytoplasm, filled with a dense layer, vesicles and membranes, extends from its distal surface along the edge of the groove, ending in a tight interlocking with another mesodermal cell. Some correlations between fine structure and the mechanics of gastrulation are discussed, and a theory of invagination is proposed, based on contraction and expansion of the dense layer and the tight junctions at distal cell surfaces.     

Tuning Plasmon Resonances for Light Coupling into Silicon: a “Rule of Thumb” for Experimental Design

For Si thin-film solar cells to become efficient, schemes to increase the optical absorption in the films are necessary. Scattering of light using plasmonic resonances in metal nanoparticles has been suggested as a feasible route. When placed on a dielectric layer on the front of a solar cell, such metal nanoparticles can scatter a large fraction of the incident light into the solar cell at the resonance wavelength, and hence increase the light collection. However, many related effects may lead to a reduction in photocurrent. Thus, nanoparticle plasmon resonances must be optimized in order to improve the overall light collection. From an experimentalist’s point of view, simple and fast experimental design tools should be explored. In this work, we investigate the plasmon-related photocurrent enhancements for Si test-solar cells with a number of different metal nanoparticle shapes and materials placed on top of a dielectric layer. The spectral position of the photocurrent-enhancement onset is compared to plasmon resonance calculations based on a fairly simple model. Despite the fact that the optical interactions in nanoparticle solar cell configurations can be quite complex, the photocurrent enhancement in the investigated test-solar cells can be predicted qualitatively well for particles with a plasmon resonance in the visible spectrum. This simple and fast model can be used as a rule of thumb in designing nanoparticle arrays for a specific photocurrent enhancement profile.     

Flexibility analysis of a surface mount technology electronic assembly plant: An integrated model using simulation

This paper focuses on developing an integrated model using simulation to evaluate the effect of several independent variables on the performance of a surface mount technology (SMT) production line. Real data and an existing SMT line from a high product mix/low volume electronics manufacturer are used to conduct the analysis. The independent variables used are set-up formation policies (group technology based family grouping methods), machine feeder types, similarity factor in set-up formation, parts reduction at design step of products, and inter-families and intra-family scheduling rules. In addition, a new method of grouping products is proposed. The measures of performance evaluated by the model are average lead time, average work-in-process (WIP) inventory and average set-up time. Data analysis shows that the proposed method of grouping products will reduce set-up time and lead time while slightly increasing WIP. The proposed simulation model helps assess the effects of some of the independent variables on line performance. Recommendations are made in order to help the user choose the best alternative to improve production line productivity and flexibility.     

Morphology of fibroblasts in collagen gels: a study using 400 keV electron microscopy and computer graphics

We have used 400 kilovolt intermediate voltage electron microscopy (IVEM) to examine thick sections of fibroblasts cultured in collagen gels. In these 3D collagen lattices, the long, narrow pseudopodial extensions that extend out and make contact with the collagen matrix exhibit a complex topography not seen in the processes put out by cells moving on planar substrata. For this reason, sections 1 to 2 microns thick that enclose a whole cell process are more informative of the overall morphology of the interaction between cells and the collagen than are thin sections. To aid the discrimination of topography of cell processes in stereo views of micrographs, some cells were labeled with antibodies and protein A-colloidal gold conjugates. The gold particles provided clear 3D reference points for computer-aided reconstructions of membrane topography from tilt series of IVEM images. Our results confirm that cells that move through collagen lattices lack the well-spread morphology of their counterparts moving on glass. They are generally rather spindly with several long branching anterior pseudopodia. We found that the cell bodies and major pseudopodial processes were cylindrical, as one might expect of cells in a 3D environment, but at the leading edge of advancing pseudopodia there are small flat extensions similar to those seen in cells on glass. This similarity suggests that the lamellipodium is a basic type of protrusive structure used by fibroblasts during locomotion on all types of substratum. The flattened shape of lamellipodia may be part of the mechanism by which cells sense the orientation of fibrillar extracellular matrices during embryonic morphogenesis.     

Isolation of separate apical,lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.     

Coupling field theory with continuum mechanics: a simulation of domain formation in giant unilamellar vesicles

Domain formation is modeled on the surface of giant unilamellar vesicles using a Landau field theory model for phase coexistence coupled to elastic deformation mechanics (e.g., membrane curvature). Smooth particle applied mechanics, a form of smoothed particle continuum mechanics, is used to solve either the time-dependent Landau-Ginzburg or Cahn-Hilliard free-energy models for the composition dynamics. At the same time, the underlying elastic membrane is modeled using smooth particle applied mechanics, resulting in a unified computational scheme capable of treating the response of the composition fields to arbitrary deformations of the vesicle and vice versa. The results indicate that curvature coupling, along with the field theory model for composition free energy, gives domain formations that are correlated with surface defects on the vesicle. In the case that external deformations are included, the domain structures are seen to respond to such deformations. The present simulation capability provides a significant step forward toward the simulation of realistic cellular membrane processes.