In JoVE (1)

Other Publications (26)

Articles by Jeffrey S. Urbach in JoVE

 JoVE Biology

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

1Department of Biochemistry, Georgetown University Medical Center, 2Department of Medicine, Georgetown University Medical Center, 3Department of Physics, Georgetown University Medical Center, 4Department of Oncology, Georgetown University Medical Center

JoVE 55177

Other articles by Jeffrey S. Urbach on PubMed

Forcing and Velocity Correlations in a Vibrated Granular Monolayer

Physical Review Letters. Aug, 2002  |  Pubmed ID: 12190468

The role of forcing on the dynamics of a vertically shaken granular monolayer is investigated. Using a flat plate, surprising negative velocity correlations are measured. A mechanism for this anticorrelation is proposed with support from both experimental results and molecular dynamics simulations. Using a rough plate, velocity correlations are positive, and the velocity distribution evolves from a Gaussian at very low densities to a broader distribution at high densities. These results are interpreted as a balance between stochastic forcing, interparticle collisions, and friction with the plate.

A New Chemotaxis Assay Shows the Extreme Sensitivity of Axons to Molecular Gradients

Nature Neuroscience. Jun, 2004  |  Pubmed ID: 15162167

Axonal chemotaxis is believed to be important in wiring up the developing and regenerating nervous system, but little is known about how axons actually respond to molecular gradients. We report a new quantitative assay that allows the long-term response of axons to gradients of known and controllable shape to be examined in a three-dimensional gel. Using this assay, we show that axons may be nature's most-sensitive gradient detectors, but this sensitivity exists only within a narrow range of ligand concentrations. This assay should also be applicable to other biological processes that are controlled by molecular gradients, such as cell migration and morphogenesis.

Predicting Axonal Response to Molecular Gradients with a Computational Model of Filopodial Dynamics

Neural Computation. Nov, 2004  |  Pubmed ID: 15476599

Axons are often guided to their targets in the developing nervous system by attractive or repulsive molecular concentration gradients. We propose a computational model for gradient sensing and directed movement of the growth cone mediated by filopodia. We show that relatively simple mechanisms are sufficient to generate realistic trajectories for both the short-term response of axons to steep gradients and the long-term response of axons to shallow gradients. The model makes testable predictions for axonal response to attractive and repulsive gradients of different concentrations and steepness, the size of the intracellular amplification of the gradient signal, and the differences in intracellular signaling required for repulsive versus attractive turning.

Introduction: First Annual Gallery of Nonlinear Images (montreal, Quebec, Canada, 2004)

Chaos (Woodbury, N.Y.). Dec, 2004  |  Pubmed ID: 15568899

Nonequilibrium Two-phase Coexistence in a Confined Granular Layer

Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics. Nov, 2004  |  Pubmed ID: 15600577

We report the observation of the homogenous nucleation of crystals in a dense layer of steel spheres confined between two horizontal plates vibrated vertically. Above a critical vibration amplitude, two-layer crystals with square symmetry were found to coexist in steady state with a surrounding granular liquid. By analogy to equilibrium hard-sphere systems, the phase behavior may be explained through entropy maximization. However, dramatic nonequilibrium effects are present, including a significant difference in the granular temperatures of the two phases.

Adaptation is Not Required to Explain the Long-term Response of Axons to Molecular Gradients

Development (Cambridge, England). Oct, 2005  |  Pubmed ID: 16176951

It has been suggested that growth cones navigating through the developing nervous system might display adaptation, so that their response to gradient signals is conserved over wide variations in ligand concentration. Recently however, a new chemotaxis assay that allows the effect of gradient parameters on axonal trajectories to be finely varied has revealed a decline in gradient sensitivity on either side of an optimal concentration. We show that this behavior can be quantitatively reproduced with a computational model of axonal chemotaxis that does not employ explicit adaptation. Two crucial components of this model required to reproduce the observed sensitivity are spatial and temporal averaging. These can be interpreted as corresponding, respectively, to the spatial spread of signaling effects downstream from receptor binding, and to the finite time over which these signaling effects decay. For spatial averaging, the model predicts that an effective range of roughly one-third of the extent of the growth cone is optimal for detecting small gradient signals. For temporal decay, a timescale of about 3 minutes is required for the model to reproduce the experimentally observed sensitivity.

Spatially Resolved Fluorescence Correlation Spectroscopy Using a Spinning Disk Confocal Microscope

Biophysical Journal. Dec, 2006  |  Pubmed ID: 16950838

We develop an extension of fluorescence correlation spectroscopy (FCS) using a spinning disk confocal microscope. This approach can spatially map diffusion coefficients or flow velocities at up to approximately 10(5) independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D~10(-7) cm(2)/s or smaller. This speed is adequate to measure small microspheres (200-nm diameter) diffusing in water, or hindered diffusion of macromolecules in complex media (e.g., tumors, cell nuclei, or the extracellular matrix). There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, has the potential for significantly higher speed at high spatial resolution. We show how to account for a pixel size effect encountered with spinning disk confocal FCS that is not present in standard or scanning FCS, and we introduce a new method to correct for photobleaching. Finally, we apply spinning disk confocal FCS to microspheres diffusing in Type I collagen, which show complex spatially varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.

Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 1. Quantification of Hemozoin Development for Drug Sensitive Versus Resistant Malaria

Biochemistry. Oct, 2006  |  Pubmed ID: 17029396

We have customized a Nipkow spinning disk confocal microscope (SDCM) to acquire three-dimensional (3D) versus time data for live, intraerythrocytic malarial parasites. Since live parasites wiggle within red blood cells, conventional laser scanning confocal microscopy produces blurred 3D images after reconstruction of z stack data. In contrast, since SDCM data sets at high x, y, and z resolution can be acquired in hundreds of milliseconds, key aspects of live parasite cellular biochemistry can be much better resolved on physiologically meaningful times scales. In this paper, we present the first 3D DIC transmittance "z stack" images of live malarial parasites and use those to quantify hemozoin (Hz) produced within the living parasite digestive vacuole, under physiologic conditions. Using live synchronized cultures and voxel analysis of sharpened DIC z stacks, we present the first quantitative in vivo analysis of the rate of Hz growth for chloroquine sensitive (CQS) versus resistant (CQR) malarial parasites. We present data for laboratory strains, as well as pfcrt transfectants expressing a CQR conferring mutant pfcrt gene. We also analyze the rate of Hz growth in the presence and absence of physiologically relevant doses of chloroquine (CQ) and verapamil (VPL) and thereby present the first in vivo quantification of key predictions from the well-known Fitch hypothesis for CQ pharmacology. In the following paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12411-12423], we acquire fluorescent images of live parasite DV via SDCM and use those to quantify DV volume for CQS versus CQR parasites.

Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 2. Altered Vacuolar Volume Regulation in Drug Resistant Malaria

Biochemistry. Oct, 2006  |  Pubmed ID: 17029397

In the previous paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12400-12410], we reported on a customized Nipkow spinning disk confocal microscopy (SDCM) system and its initial application to DIC imaging of hemozoin within live, synchronized, intraerythrocytic Plasmodium falciparum malarial parasites. In this paper, we probe the biogenesis as well as the volume and pH regulation of the parasite digestive vacuole (DV), using the fluorescence imaging capabilities of the system. Several previous reports have suggested that mutant PfCRT protein, which causes chloroquine resistance (CQR) in P. falciparum, also causes increased acidification of the DV. Since pH and volume regulation are often linked, we wondered whether DV volume differences might be associated with CQR. Using fast acquisition of SDCM z stacks for synchronized parasites with OGd internalized into the DV, followed by iterative deconvolution using experimental point spread functions, we quantify the volume of the DV for live, intraerythrocytic HB3 (CQS), Dd2 (CQR via drug selection), GCO3 (CQS), and GCO3/C3(Dd2) (CQR via transfection with mutant pfcrt) malarial parasites as they develop within the human red blood cell. We find that relative to both CQS strains, both CQR strains show significantly increased DV volume as the organelle forms upon entry into the trophozoite stage of development and that this persists until the trophozoite-schizont boundary. A more acidic DV pH is found for CQR parasites as soon as the organelle forms and persists throughout the trophozoite stage. We probe DV volume and pH changes upon ATP depletion, hypo- and hypertonic shock, and rapid withdrawal of perfusate chloride. Taken together, these data suggest that the PfCRT mutations that cause CQR also lead to altered DV volume regulation.

Depletion Force in a Bidisperse Granular Layer

Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics. Nov, 2007  |  Pubmed ID: 18233652

We demonstrate the effect of the depletion force in experiments and simulations of vertically vibrated mixtures of large and small steel spheres. The system exhibits size segregation and a large increase in the pair correlation function of the large spheres for short distances that can be accurately described using a combination of the depletion potential derived for equilibrium colloidal systems and a Boltzmann factor. The Boltzmann factor defines an effective temperature for the system, which we compare to other measures of temperature.

Design and Optimization of a High-speed, High-sensitivity, Spinning Disk Confocal Microscopy System

Journal of Biomedical Optics. Sep-Oct, 2008  |  Pubmed ID: 19021437

We describe the principles, design, and systems integration of a flexible, high-speed, high-sensitivity, high-resolution confocal spinning disk microscopy (SDCM) system. We present several artifacts unique to high-speed SDCM along with techniques to minimize them. We show example experimental results from a specific implementation capable of generating 3-D image stacks containing 30 2-D slices at 30 stacks per second. This implementation also includes optics for differential interference contrast (DIC), phase, and bright-field imaging, as well as an optical trap with sensitive force and position measurement.

Effect of Inelasticity on the Phase Transitions of a Thin Vibrated Granular Layer

Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics. Nov, 2008  |  Pubmed ID: 19113119

We describe an experimental and computational investigation of the ordered and disordered phases of a vibrating thin, dense granular layer composed of identical metal spheres. We compare the results from spheres with different amounts of inelasticity and show that inelasticity has a strong effect on the phase diagram. We also report the melting of an ordered phase to a homogeneous disordered liquid phase at high vibration amplitude or at large inelasticities. Our results show that dissipation has a strong effect on ordering and that in this system ordered phases are absent entirely in highly inelastic materials.

Optical Neuronal Guidance in Three-dimensional Matrices

Journal of Neuroscience Methods. May, 2009  |  Pubmed ID: 19428538

We demonstrate effective guidance of neurites extending from PC12 cells in a three-dimensional collagen matrix using a focused infrared laser. Processes can be redirected in an arbitrarily chosen direction in the imaging plane in approximately 30 min with an 80% success rate. In addition, the application of the laser beam significantly increases the rate of neurite outgrowth. These results extend previous observations on 2D coated glass coverslips. We find that the morphology of growth cones is very different in 3D than in 2D, and that this difference suggests that the filopodia play a key role in optical guidance. This powerful, flexible, non-contact guidance technique has potentially broad applications in tissues and engineered environments.

Chloroquine Transport in Plasmodium Falciparum. 1. Influx and Efflux Kinetics for Live Trophozoite Parasites Using a Novel Fluorescent Chloroquine Probe

Biochemistry. Oct, 2009  |  Pubmed ID: 19728740

Several models for how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Distinguishing between these models requires detailed analysis of high-resolution CQ transport data that is unfortunately impossible to obtain with traditional radio-tracer methods. Thus, we have designed and synthesized fluorescent CQ analogues for drug transport studies. One probe places a NBD (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid) group at the tertiary aliphatic N of CQ, via a flexible 6 C amide linker. This probe localizes to the malarial parasite digestive vacuole (DV) during initial perfusion under physiologic conditions and exhibits similar pharmacology relative to CQ, vs both CQ-sensitive (CQS) and CQ-resistant (CQR) parasites. Using live, synchronized intraerythrocytic parasites under continuous perfusion, we define NBD-CQ influx and efflux kinetics for CQS vs CQR parasites. Since this fluorescence approach provides data at much higher kinetic resolution relative to fast-filtration methods using (3)H-CQ, rate constants vs linear initial rates for CQ probe flux can be analyzed in detail. Importantly, we find that CQR parasites have a decreased rate constant for CQ influx into the DV and that this is due to mutation of PfCRT. Analysis of zero trans efflux for CQS and CQR parasites suggests that distinguishing between bound vs free pools of intra-DV drug probe is essential for proper kinetic analysis of efflux. The accompanying paper (DOI 10.1021/bi901035j ) further probes efflux kinetics for proteoliposomes containing purified, reconstituted PfCRT.

Quantitative Studies of Neuronal Chemotaxis in 3D

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19763971

During development a variety of cell types are guided by molecular concentration gradients to form tissues and organ systems. In the nervous system, the migration and neuronal pathfinding that occurs during development is organized and driven by "guidance cues." Some of these cues are substrate bound or nondiffusible, while many are diffusible and form gradients within the developing embryo to guide neurons and neurites to their appropriate destination. There have been many approaches used to discover and characterize the multitude of guidance cues, their cognate receptors, and how these cues and receptors are regulated to achieve the highly detailed connections found in the nervous system. Here we present a method for creating precisely controlled gradients of molecular factors within a three-dimensional culture environment. The method is based on a non contact mediated delivery of biomolecules to the surface of a collagen gel. The factors are printed in a pattern on the top of a gel containing the tissue or cell type of interest embedded in the gel. The formation of the gradient is dependent upon the diffusion of the printed molecule in the gel. The concentration of the factor within the gel becomes independent of depth rapidly, and the gradient becomes smooth on a similar time scale. The gradients formed can remain relatively stable for a day or more. Moreover, the steepness and molar concentration of tropic or trophic factors within the gradient can be controlled.

Event Ordering in Live-cell Imaging Determined from Temporal Cross-correlation Asymmetry

Biophysical Journal. Jun, 2010  |  Pubmed ID: 20513386

We use the temporal asymmetry of the cross-correlation function to determine the temporal ordering of spatially localized cellular events in live-cell multichannel fluorescence imaging. The analysis is well suited to noisy, stochastic systems where the temporal order may not be apparent in the raw data. The approach is applicable to any biochemical reaction not in chemical equilibrium, including protein complex assembly, sequential enzymatic processes, gene regulation, and other cellular signaling events. As an automated quantitative measure, this approach allows the data to be readily interpreted statistically with minimal subjective biases. We first test the technique using simulations of simple biophysical models with a definite temporal ordering. We then demonstrate the approach by extracting the temporal ordering of three proteins-actin, sorting nexin 9, and clathrin-in the endocytic pathway.

Size-dependent Rheology of Type-I Collagen Networks

Biophysical Journal. Oct, 2010  |  Pubmed ID: 20959077

We investigate the system size-dependent rheological response of branched type I collagen gels. When subjected to a shear strain, the highly interconnected mesh dynamically reorients, resulting in overall stiffening of the network. When a continuous shear strain is applied to a collagen network, we observe that the local apparent modulus, in the strain-stiffening regime, is strongly dependent on the gel thickness. In addition, we demonstrate that the overall network failure is determined by the ratio of the gel thickness to the mesh size. These findings have broad implications for cell-matrix interactions, the interpretation of rheological tissue data, and the engineering of biomimetic scaffolds.

Four-dimensional Structural Dynamics of Sheared Collagen Networks

Chaos (Woodbury, N.Y.). Dec, 2011  |  Pubmed ID: 22225332

Strength in the Periphery: Growth Cone Biomechanics and Substrate Rigidity Response in Peripheral and Central Nervous System Neurons

Biophysical Journal. Feb, 2012  |  Pubmed ID: 22325267

There is now considerable evidence of the importance of mechanical cues in neuronal development and regeneration. Motivated by the difference in the mechanical properties of the tissue environment between the peripheral (PNS) and central (CNS) nervous systems, we compare substrate-stiffness-dependent outgrowth and traction forces from PNS (dorsal root ganglion (DRG)) and CNS (hippocampal) neurons. We show that neurites from DRG neurons display maximal outgrowth on substrates with a Young's modulus of ∼1000 Pa, whereas hippocampal neurite outgrowth is independent of substrate stiffness. Using traction force microscopy, we also find a substantial difference in growth cone traction force generation, with DRG growth cones exerting severalfold larger forces compared with hippocampal growth cones. The traction forces generated by DRG and hippocampal growth cones both increase with increasing stiffness, and DRG growth cones growing on substrates with a Young's modulus of 1000 Pa strengthen considerably after 18-30 h. Finally, we find that retrograde actin flow is almost three times faster in hippocampal growth cones than in DRG. Moreover, the density of paxillin puncta is significantly lower in hippocampal growth cones, suggesting that stronger substrate coupling of the DRG cytoskeleton is responsible for the remarkable difference in traction force generation. These findings reveal a differential adaptation of cytoskeletal dynamics to substrate stiffness in growth cones of different neuronal types, and highlight the potential importance of the mechanical properties of the cellular environment for neuronal navigation during embryonic development and nerve regeneration.

Optical and Physicochemical Characterization of the Luminous Mucous Secreted by the Marine Worm Chaetopterus Sp

Physiological and Biochemical Zoology : PBZ. Nov-Dec, 2013  |  Pubmed ID: 24241067

Bioluminescence of the marine worm Chaetopterus variopedatus was first investigated several decades ago mainly using tissue extract. Light production of the worm, however, originates from a secreted mucus only. Here, we report the optical and physicochemical properties of the luminous mucus. We show that the produced light occurs as a long glow in the blue range (455 nm), which is an unusual color for a shallow benthic invertebrate. We also show that the light originates from a photoprotein whose light production is independent of molecular oxygen yet somewhat related to the physicochemical (rheological) characteristics of the mucus itself. Indeed, the mucus seems to polymerize and become more viscous on exposure to H2O2, which in turn seems to inhibit the light production. Ferrous iron was not associated with any strong stimulatory effect. This is in contrast to past studies on worm tissues showing that the light production is strongly stimulated by H2O2 and ferrous iron. Overall, our results highlight the fact that working on the luminous mucus only (vs. worm tissues) provides the ability to study its chemical properties possibly involved in the fine control of light production-as well as its rheological properties-and identify the possible interactions between these two properties.

The Effects of Confinement on Neuronal Growth Cone Morphology and Velocity

Biomaterials. Aug, 2014  |  Pubmed ID: 24840617

Optimizing growth cone guidance through the use of patterned substrates is important for designing regenerative substrates to aid in recovery from neuronal injury. Using laser ablation, we designed micron-scale patterns capable of confining dissociated mouse cerebellar granule neuron growth cones to channels of different widths ranging from 1.5 to 12 μm. Growth cone dynamics in these channels were observed using time-lapse microscopy. Growth cone area was decreased in channels between 1.5 and 6 μm as compared to that in 12 μm and unpatterned substrates. Growth cone aspect ratio was also affected as narrower channels forced growth cones into a narrow, elongated shape. There was no difference in the overall rate of growth cone advance in uniform channels between 1.5 and 12 μm as compared to growth on unpatterned substrates. The percentage of time growth cones advanced, paused, and retracted was also similar. However, growth cones did respond to changes in confinement: growth cones in narrow lanes rapidly sped up when encountering a wide region and then slowed down as they entered another narrow region. Our results suggest that the rate of neurite extension is not affected by the degree of confinement, but does respond to changes in confinement.

Shear-driven Aggregation of SU-8 Microrods in Suspension

Soft Matter. Sep, 2014  |  Pubmed ID: 24975104

A non-Brownian suspension of micron scale rods exhibits reversible shear-driven formation of disordered aggregates resulting in dramatic viscosity enhancement at low shear rates. Aggregate formation is imaged using a combined rheometer and fluorescence microscope. The size and structure of these aggregates are found to be a function of shear rate and concentration, with larger aggregates present at lower shear rates and higher concentrations. Quantitative measurements of the early-stage aggregation process are modeled by collision driven growth of porous structures which suggest that the aggregate density increases with shear rate. This result is combined with a Krieger-Dougherty type constitutive relationship and steady-state viscosity measurements to estimate the intrinsic viscosity of complex structures developed under shear. These results represent a direct, quantitative, experimental demonstration of the association between aggregation and viscosity enhancement for a rod suspension, and demonstrate a way of inferring microscopic geometric properties of a dynamic system through the combination of quantitative imaging and rheology.

Stress Heterogeneities in Sheared Type-I Collagen Networks Revealed by Boundary Stress Microscopy

PloS One. 2015  |  Pubmed ID: 25734484

Disordered fiber networks provide structural support to a wide range of important materials, and the combination of spatial and dynamic complexity may produce large inhomogeneities in mechanical properties, an effect that is largely unexplored experimentally. In this work, we introduce Boundary Stress Microscopy to quantify the non-uniform surface stresses in sheared collagen gels. We find local stresses exceeding average stresses by an order of magnitude, with variations over length scales much larger than the network mesh size. The strain stiffening behavior observed over a wide range of network mesh sizes can be parameterized by a single characteristic strain and associated stress, which describes both the strain stiffening regime and network yielding. The characteristic stress is approximately proportional to network density, but the peak boundary stress at both the characteristic strain and at yielding are remarkably insensitive to concentration.

Adaptation is Not Required to Explain the Long-term Response of Axons to Molecular Gradients

Development (Cambridge, England). Jul, 2015  |  Pubmed ID: 26130758

Traction Force and Tension Fluctuations in Growing Axons

Frontiers in Cellular Neuroscience. 2015  |  Pubmed ID: 26578882

Actively generated mechanical forces play a central role in axon growth and guidance, but the mechanisms that underly force generation and regulation in growing axons remain poorly understood. We report measurements of the dynamics of traction stresses from growth cones of actively advancing axons from postnatal rat DRG neurons. By tracking the movement of the growth cone and analyzing the traction stress field from a reference frame that moves with it, we are able to show that there is a clear and consistent average stress field that underlies the complex spatial stresses present at any one time. The average stress field has strong maxima on the sides of the growth cone, directed inward toward the growth cone neck. This pattern represents a contractile stress contained within the growth cone, and a net force that is balanced by the axon tension. Using high time-resolution measurements of the growth cone traction stresses, we show that the stress field is composed of fluctuating local stress peaks, with a large number peaks that live for a short time, a population of peaks whose lifetime distribution follows an exponential decay, and a small number of very long-lived peaks. We show that the high time-resolution data also reveal that the tension appears to vary randomly over short time scales, roughly consistent with the lifetime of the stress peaks, suggesting that the tension fluctuations originate from stochastic adhesion dynamics.

Torsional Stiffness Determines Aggregate Structure in Sheared Colloidal Rod Suspensions

Soft Matter. Sep, 2016  |  Pubmed ID: 27714292

We report the results of simulations of suspensions of sheared rigid rods in the presence of strong attractive inter-particle interactions, using dissipative particle dynamics for a coarse-grained representation of the suspending fluid. We find that the combined effect of the attractive interactions and shear-induced alignment generically produces aggregates of aligned bundles when the contact points between rods are free to rotate. However, the introduction of substantial torsional stiffness to the inter-particle contacts recapitulates the disordered aggregates often observed in suspensions of high aspect ratio particles. We show that the degree of alignment within the aggregates depends on the strength of the torsional stiffness, while the stability of the aggregates and their impact on system viscosity depend on the competition between the attractive interaction and the shear stresses.

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