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PubMed2004

Elastic behavior of a two-dimensional crystal near melting.

Using positional data from video microscopy, we determine the elastic moduli of two-dimensional colloidal crystals as a function of temperature. The moduli are extracted from the wave-vector-dependent normal-mode spring constants in the limit q-->0 and are compared to the renormalized Young's modulus of the Kosterlitz-Thouless-Halperin-Nelson-Young theory. An essential element of this theory is the universal prediction that Young's modulus must approach 16 pi at the melting temperature. This is indeed observed in our experiment.

Journal Article↗
PubMed2005

Capillary interactions between anisotropic colloidal particles.

We report on the behavior of micron-sized prolate ellipsoids trapped at an oil-water interface. The particles experience strong, anisotropic, and long-ranged attractive capillary interactions which greatly exceed the thermal energy k(B)T. Depending on surface chemistry, the particles aggregate into open structures or chains. Using video microscopy, we extract the pair interaction potential between ellipsoids and show it exhibits a power law behavior over the length scales probed. Our observations can be explained using recent calculations, if we describe the interfacial ellipsoids as capillary quadrupoles.

Anisotropy↗
PubMed2005

Pair interaction of dislocations in two-dimensional crystals.

The pair interaction between crystal dislocations is systematically explored by analyzing particle trajectories of two-dimensional colloidal crystals measured by video microscopy. The resulting pair energies are compared to Monte Carlo data and to predictions derived from the standard Hamiltonian of the elastic theory of dislocations. Good agreement is found with respect to the distance and temperature dependence of the interaction potential, but not regarding the angle dependence where discrete lattice effects become important. Our results on the whole confirm that the dislocation Hamiltonian allows a quantitative understanding of the formation and interaction energies of dislocations in two-dimensional crystals.

Journal Article↗
PubMed2006

Two scenarios for colloidal phase transitions.

A two-dimensional assembly of charged colloidal particles induced by an alternating electric field was studied in real space by means of digital video microscopy. Phase transitions occur from a highly ordered colloidal monolayer to an isotropic suspension by changing the field strength or frequency (in the appropriate range). In particular, it is found that the strength-dependent phase transition is an infinite-order phase transition, in contrast with the frequency-dependent phase transition, which is a second-order phase transition.

Journal Article↗
PubMed2006

Multipoint holographic optical velocimetry in microfluidic systems.

We show how holographic optical trapping can be used for the multipoint measurement of fluid flow in microscopic geometries. An array of microprobes can be simultaneously trapped and used to map out the fluid flow in a microfluidic device. The optical traps are alternately turned on and off such that the probe particles are displaced by the flow of the surrounding fluid and then retrapped. The particles' displacements are monitored by digital video microscopy and directly converted into velocity field values. This technique enables the measurement of a two-dimensional flow field at points arbitrarily distributed in a three-dimensional volume. The validity of the technique is demonstrated for the case of the flow around a spinning sphere and the flow at the outlet of a microchannel.

Journal Article↗
PubMed2006

Dynamics of individual single-walled carbon nanotubes in water by real-time visualization.

Individual single-walled carbon nanotubes (SWNTs) in aqueous suspension are visualized directly by fluorescence video microscopy. The fluorescent tagging is simple, biocompatible, and does not modify the SWNTs. The dynamics of individual SWNTs in water are observed and quantified for the first time. We measure the confined rotational diffusion coefficient and find it in reasonable agreement with predictions based on confined diffusion of dilute Brownian rods. We determine the critical concentration at which SWNTs in suspensions start interacting. By analyzing the fluctuating shape of SWNTs in the 3 to 5 microm range, we determine that their persistence length ranges between 32 and 174 microm, in agreement with theoretical estimates; thus, commonly available SWNTs in liquids can be considered as rigid Brownian rods in the absence of imposed external fields or self-attractive forces.

Journal Article↗
PubMed1997

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (II. 2,6-Dichlorobenzonitrile Inhibition of Motility and Stalk Production in the Marine Diatom Achnanthes longipes).

The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) and the DCB analogs 2-chloro-6-fluorobenzonitrile, 3-amino-2,6-dichlorobenzonitrile, and 5-dimethylamino-naphthalene-1-sulfonyl-(3-cyano-2, 4-dichloro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanthes longipes, resulting in a loss of motility and a lack of permanent adhesion. The effect was fully reversible upon removal of the inhibitor, and cell growth was not affected at concentrations of inhibitors adequate to effectively interrupt the adhesion sequence. Video microscopy revealed that the adhesion sequence was mediated by the export and assembly of polymers, and consisted of initial attachment followed by cell motility and eventual production of permanent adhesive structures in the form of stalks that elevated the diatom above the substratum. A. longipes adhesive polymers are primarily composed of noncellulosic polysaccharides (B.A. Wustman, M.R. Gretz, and K.D. Hoagland [1997] Plant Physiol 113: 1059-1069). These results, together with the discovery of DCB inhibition of extracellular matrix assembly in noncellulosic red algal unicells (S.M. Arad, O. Dubinsky, and B. Simon [1994] Phycologia 33: 158-162), indicate that DCB inhibits synthesis of noncellulosic extracellular polysaccharides. A fluorescent probe, DCBF, was synthesized and shown to inhibit adhesive polymer production in the same manner as DCB. DCBF specifically labeled an 18-kD polypeptide isolated from a membrane fraction. Inhibition of adhesion by DCB and its analogs provides evidence of a direct relationship between polysaccharide synthesis and motility and permanent adhesion.

Journal Article↗
PubMed1994

Investigations on protein crystal growth by the gel acupuncture method.

In this work we explore the possibilities of the gel-acupuncture technique, proposed previously for the growth of protein single crystals [García-Ruiz, Moreno, Viedma & Coll (1993). Mater. Res. Bull. 28, 541-546]. The main advantage of the technique is that the crystals are obtained inside an X-ray capillary and, unlike classical microdiffusion techniques, it involves a very simple and accurate technical arrangement that permits the continuous monitoring of the crystals in their growth environment. In particular, we describe the growth of single crystals of lysozyme, concanavalin A and ribonuclease A. Different starting conditions have been used to grow single crystals of these proteins into different types of capillaries at several protein and precipitating-agent concentrations. It is demonstrated that the technique works for a wide range of precipitating agents commonly used in protein crystal growth, such as large polymers (PEG 4000 and PEG 6000), organic solvents (from methanol to butanol) and salts [NaCl, (NH(4))(2)SO(4)]. The range of inner diameter of the capillaries for which the technique works correctly has been also studied. The growth process and possible crystal movement was followed by video microscopy. Lysozyme crystals up to 3.1 mm were obtained but the average maximum linear crystal sizes were 2.0 mm for lysozyme, 0.4 mm for concanavalin A and 1.2 mm for ribonuclease, respectively. The waiting times to reach such a size, measured from the set-up of the experiments, were 72 h, 10 d and 5 d, respectively. Gels of tetramethoxysilane, tetraethoxysilane, sodium silicate, agar, high-strength agar and gel-gro have been tested in relation to their mechanical properties and their chemical interaction with the reactants. Finally, we discuss briefly the advantages of the gel-acupuncture technique and plausible applications other than crystal growth.

Journal Article↗
PubMed2005

Influence of lipid shell physicochemical properties on ultrasound-induced microbubble destruction.

We present the first study of the effects of monolayer shell physicochemical properties on the destruction of lipid-coated microbubbles during insonification with single, one-cycle pulses at 2.25 MHz and low-duty cycles. Shell cohesiveness was changed by varying phospholipid and emulsifier composition, and shell microstructure was controlled by postproduction processing. Individual microbubbles with initial resting diameters between 1 and 10 microm were isolated and recorded during pulsing with bright-field and fluorescence video microscopy. Microbubble destruction occurred through two modes: acoustic dissolution at 400 and 600 kPa and fragmentation at 800 kPa peak negative pressure. Lipid composition significantly impacted the acoustic dissolution rate, fragmentation propensity, and mechanism of excess lipid shedding. Less cohesive shells resulted in micron-scale or smaller particles of excess lipid material that shed either spontaneously or on the next pulse. Conversely, more cohesive shells resulted in the buildup of shell-associated lipid strands and globular aggregates of several microns in size; the latter showed a significant increase in total shell surface area and lability. Lipid-coated microbubbles were observed to reach a stable size over many pulses at intermediate acoustic pressures. Observations of shell microstructure between pulses allowed interpretation of the state of the shell during oscillation. We briefly discuss the implications of these results for therapeutic and diagnostic applications involving lipid-coated microbubbles as ultrasound contrast agents and drug/gene delivery vehicles.

Coated Materials, Biocompatible↗
PubMed1996

Imposition of a physiologic DC electric field alters the migratory response of human keratinocytes on extracellular matrix molecules.

Outwardly directed ionic currents have been measured leaving skin wounds in vivo. These currents generate physiologic electric fields of approximately 100 mV/mm, which may function to direct keratinocyte migration toward the healing wound. We investigated whether the substrate on which the keratinocyte migrates modulates the galvanotactic response to an electric migratory signal. Cultured human keratinocytes were plated on different matrices; types I and IV collagen, fibronectin, laminin, and tissue culture plastic. The effect of an applied direct current (DC) electric field on directional migration was monitored by time-lapse video microscopy over a 2-h period. Directionality was quantitated by calculating the cosine of the angle of migration in relation to anodal-cathodal orientation. Migration toward the negative pole was observed on all matrices as compared with controls (no applied field), which displayed random migration. No significant increase in directional response occurred when the field strength was increased by 100 mV/mm (physiologic levels) to 400 mV/mm. The degree of directionality and the average net cell translocation however, varied significantly with the substrate. The greatest cathodal migration in response to a DC electric field was observed with keratinocytes plated on types I and IV collagens and plastic. The directional migratory response was least on a laminin substrate, whereas cells on fibronectin demonstrated a response that was intermediate between those of collagen and laminin. These results suggest that physiologic ionic currents in concert with underlying matrix may influence the rate of reepithelialization of skin wounds.

Cell Movement↗
PubMed1993

Migration of human melanoma cells on hyaluronate is related to CD44 expression.

Phenotypic and functional aspects of melanoma-hyaluronate interactions were investigated by studying the expression of CD44, cell migration, and transmembrane penetration of human melanoma cell lines on hyaluronate-coated substrates. Expression of CD44 was tested by flow cytometry on seven human melanoma cell lines. Strong reactivity with anti-CD44 monoclonal antibody was observed in four of seven of the cell lines. Migration studies of CD44(+) cell lines on hyaluronic acid- and chondroitin-6-sulfate-coated substrates, using time-lapse video-microscopy, showed a dramatic dose-dependent increase in migration rate on hyaluronate but not on chondroitin-6-sulfate. Moreover, CD44(-) cell lines showed no modification in migration rate on either substrate. Addition of soluble hyaluronate produced a dose-dependent inhibition of acceleration of CD44(+)cells on hyaluronate-coated substrates, whereas addition of chondroitin-6-sulfate had no effect. Migration inhibition experiments with soluble CD44 (CD44 receptor globulin) also showed specific blocking of the migration of CD44(+) cells on hyaluronate. Haptotactic invasion was increased in CD44(+) cell lines through hyaluronate-coated polycarbonate membranes, whereas no change was detected on chondroitin-6-sulfate-coated membranes. CD44(-) cell lines showed no response to either type of coating. In the melanoma cell lines tested, the expression of CD44 correlated with in vitro migration and invasiveness on hyaluronate substrates. Taken together, our data are consistent with the suggestion that CD44 may play a role in stimulating in vivo aggressiveness of tumors through hyaluronate-rich stroma.

Cell Movement↗
PubMed2003

Mechanism of mitochondrial dysfunction in diabetic sensory neuropathy.

Symmetrical sensory polyneuropathy, the most common form of diabetic neuropathy in humans, is associated with a spectrum of structural changes in peripheral nerve that includes axonal degeneration, paranodal demyelination, and loss of myelinated fibers--the latter probably the result of a dying-back of distal axons. Mitochondrial dysfunction has recently been proposed as an etiological factor in this degenerative disease of the peripheral nervous system. Lack of neurotrophic support has been proposed as a contributing factor in the etiology of diabetic neuropathy based on studies in animal models of Type I diabetes. We have recently demonstrated that insulin and neurotrophin-3 (NT-3) modulate mitochondrial membrane potential in cultured adult sensory neurons. We therefore tested the hypothesis that diabetes-induced mitochondrial dysfunction is caused by impairments in neurotrophic support. We have used real-time fluorescence video microscopy to analyze mitochondrial membrane potential in cultured adult sensory neurons isolated from normal and diabetic rats. Diabetes caused a significant loss of mitochondrial membrane potential in all sub-populations of sensory neurons which can be prevented by in vivo treatment with insulin or NT-3. The mechanism of insulin and NT-3-dependent modulation of mitochondrial membrane potential involves the activation of the phosphoinositide 3 kinase (PI 3 kinase) pathway. Downstream targets of PI 3 kinase, such as Akt and the transcription factor cAMP response element-binding protein (CREB), are activated by insulin and NT-3 and regulate sensory neuron gene expression. These alterations in gene expression modulate critical components of metabolite pathways and the electron transport chain associated with the neuronal mitochondrion. Our results show that in adult sensory neurons, treatment with insulin can elevate the input of reducing equivalents into the mitochondrial electron transport chain, which leads to greater mitochondrial membrane polarization and enhanced ATP synthesis.

Adenosine Triphosphate↗
PubMed1993

Capillary underperfusion in chronic venous insufficiency: a cause for leg ulceration?

Dermal capillaries in the goiter area of the lower leg were examined by video-microscopy before and after the administration of intravenous fluorescein in 13 patients with chronic venous insufficiency (CVI) who were at risk of developing leg ulceration, and in 13 normal controls. The influence of posture on capillary perfusion was determined by viewing the same area of skin with the leg in both the supine and dependent positions. Capillary density was lower in patients than in controls, irrespective of the position of the leg (P < 0.01). Fluorescence angiography studies in normal controls showed a reduction in capillary density with dependency (P < 0.01), but patients with CVI showed no significant change. Fluorescence angiography revealed a greater number of capillaries than seen during native capillaroscopy (P < 0.05). The decreased capillary density, and the loss of the postural vasoconstrictor reflex in patients with chronic venous incompetence may play a role in the pathogenesis of ulceration.

Adult↗
PubMed2005

A new antihistamine levocetirizine inhibits eosinophil adhesion to vascular cell adhesion molecule-1 under flow conditions.

BACKGROUND: We previously demonstrated that low concentrations of a new antihistamine levocetirizine inhibited eosinophil transmigration through human microvascular endothelial cells. OBJECTIVE: Here, the inhibitory effect of levocetirizine on eosinophil adhesion to recombinant human vascular cell adhesion molecule-1 (rhVCAM)-1 was examined under conditions of shear stress using an in vitro model of the post-capillary venules. METHODS: Eosinophils isolated from normal subjects were pre-incubated with a concentration range of levocetirizine (10(-6)-10(-10) m) or negative dilution control. Resting or granulocyte macrophage-colony stimulating factor (GM-CSF)-stimulated cells were pumped through rhVCAM-1 (10 microg/mL) coated capillary tubes using a microfluidic syringe pump at a precise and constant flow rate (1 dyn/cm(2)). Images of rolling and firmly adherent eosinophils were captured using real-time video microscopy. RESULTS: Levocetirizine significantly inhibited resting eosinophil adhesion to rhVCAM-1 with maximal effect at 10(-8) M with an EC(50) of 10(-9) m. Levocetirizine almost abolished resting eosinophil adhesion by the 15 min time-point. GM-CSF significantly enhanced eosinophil adhesion and their ability to flatten on rhVCAM-1. Both phenomena were inhibited by levocetirizine in a dose-dependent manner, at both 5 and 15 min (optimal concentration of 10(-8) m with an EC(50) of 10(-9) m). Real-time imaging revealed that the effect of levocetirizine on post-adhesion behaviour (detachment, flatness) contributed to its inhibitory action on eosinophil adhesion to rhVCAM-1. In contrast, very late antigen (VLA)-4 mAb inhibited eosinophil adhesion to rhVCAM-1 from the earliest time-points. CONCLUSION: Physiologically relevant concentrations of levocetirizine inhibit resting and GM-CSF-stimulated firm eosinophil adhesion to rhVCAM-1 under flow conditions.

Antibodies, Monoclonal↗
PubMed1992

Morphological and cytoskeletal changes in epithelial cells occur immediately upon interaction with Salmonella typhimurium grown under low-oxygen conditions.

Salmonella typhimurium grown under oxygen-limiting conditions were found to enter into, elicit actin filament rearrangement in, and effect morphological changes upon HEp-2 cells within 15 min after infection. Video microscopy revealed that host cell morphological changes associated with entry began within 1 min of productive adherence. Polarized Caco-2 cell morphology was affected 40 s after infection with low-oxygen-grown S. typhimurium. Stationary-phase S. typhimurium did not elicit these phenomena within this time-period even when adherence was enhanced with the afimbial adhesin, AFA-I. Thus, environmental cues regulate S. typhimurium invasion factors, allowing for immediate entry into host cells. Additionally, actin filament rearrangement and morphological changes in the eukaryotic host cell are essential for entry and occur within minutes of infection.

Actins↗
PubMed1990

Characterization of an active, fluorescein-labelled kinesin.

Kinesin was isolated from bovine intradural nerve roots and conjugated with 5-(iodoacetamido)fluorescein. The modified kinesin (AF-kinesin) supports the movement of organelles along microtubules at rates comparable with those obtained using unmodified kinesin. AF-kinesin was purified by high-performance liquid chromatography. SDS/PAGE analysis of the purified fraction showed the presence of a fluorescent band at the position of the 125-kDa kinesin heavy chain. This protein promoted microtubule gliding with MgATP and with MgGTP at rates comparable to those of unlabelled kinesin. AF-kinesin had a fluorescein/protein ratio of one. Video microscopy at low light levels was used to monitor the interactions between the analogue and microtubules. AF-kinesin binds to microtubules in the presence of adenosine 5'-[beta, gamma-imino]triphosphate or ADP. Brief incubation of the microtubule. AF-kinesin complex with 10 mM ATP or GTP completely removes the labelled molecule. AF-kinesin can be inactivated in its ability to cause microtubule gliding by irradiating it with light that bleaches the bound fluorophore. When the protein is damaged in this way it still binds to microtubules and does so in the presence of ATP.

Adenosine Triphosphatases↗
PubMedDate not supplied

Development of an objective, semiautomated method for assessment of sperm motility.

Study of sperm motility is associated to the development of precise and economical systems of evaluation. The purpose of this work was to develop an Objective Semi-Automated Method (MOSA) to evaluate the sperm motility. Human semen samples were registered by video-microscopy. The same videofilms were analysed with the MOSA, the subjective method and the automated CellSoft method. The percentages of motile and immotile sperms were equivalent with the three methods. The percentages of rapidly and slowly motile sperms were similar both with the MOSA and the subjective method. The curvilinear and linear velocities as well as the linearity values obtained with the MOSA were different to those obtained with the CellSoft, although such differences would be biologically acceptable. MOSA is an inexpensive, objective and precise method that does not require trained technicians and allows evaluation of several parameters of sperm motility.

Equipment Design↗
PubMedDate not supplied

Structure and function of the adventitial and mucosal nerve plexuses of the bronchial tree in the developing lung.

1. The aim of the present study was first to determine when airway smooth muscle first appears in the airways of the developing foetal lung and when its contractility is mature and, second, when the airway smooth muscle becomes innervated, both structurally and functionally. 2. Narrowing and relaxation of the airways of the intact bronchial tree from the lungs of foetal pigs at varying stages of gestation was recorded by real time video microscopy. Nerves and smooth muscle were stained immunohistochemically and their spatial relationships were visualized by confocal microscopy. 3. From early in gestation (pseudoglandular stage/branching morphogenesis), airways were covered with a single layer of smooth muscle cells orientated cylindrically around the airway wall. Thick nerve trunks containing loosely packed nerves, with ganglia forming at their junctions ensheathed the airways to the growing tips, with a network of fine varicosed nerves lying on the smooth muscle cells. Some of these nerves were functional and cholinergic, as electrical field stimulation caused substantial narrowing that was blocked by atropine and by tetrodotoxin. By mid-term, an extensive plexus of nerves, well-defined ganglia and varicosed nerves to the smooth muscle had developed. Some nerves extended through the airway smooth muscle to form the elements of a mucosal nerve plexus in conjunction with developing vascular tissue forming a bronchial circulation. In 3 week postnatal pigs the distal and terminal airways were densely supplied with varicosed nerves to the smooth muscle. The ganglia were more centrally located. 4. An abundant network of calcitonin gene-related peptide (CGRP) nerves with prominent nerve endings lay just below the surface of the epithelium. The nerve bundles to the terminal arterioles in the mucosa also contained CGRP-positive fibres as well as sympathetic nerves (neuropeptide Y- and tyrosine hydroxylase-positive). It is hypothesized that these epithelial and arteriolar CGRP nerves form the local axon reflex purported to cause neurogenic inflammation. 5. The spontaneously active tone exerted by the airway smooth muscle from early in gestation is hypothesized to provide the force across the airway wall and adjacent parenchyma that is the stimulus for lung growth in foetal life.

Animals↗