Maria Kilfoil

Accurate detection and complete tracking of large populations of features in three dimensions.

Localization and tracking of colloidal particles in microscopy images generates the raw data necessary to understand both the dynamics and the mechanical properties of colloidal model systems. Yet, despite the obvious importance of analyzing particle movement in three dimensions (3D), accurate sub-pixel localization of the particles in 3D has received little attention so far. Tracking has been limited by the choice of whether to track all particles in a low-density system, or whether to neglect the most mobile fraction of particles in a dense system. Moreover, assertions are frequently made on the accuracies of methods for locating particles in colloid physics and in biology, and the field of particle locating and tracking can be well-served by quantitative comparison of relative performances. We show that by iterating sub-pixel localization in three dimensions, the centers of particles can be more accurately located in three-dimensions (3D) than with all previous methods by at least half an order of magnitude. In addition, we show that implementing a multi-pass deflation approach, greater fidelity can be achieved in reconstruction of trajectories, once particle positions are known. In general, all future work must defend the accuracy of the particle tracks to be considered reliable. Specifically, other researchers must use the methods presented here (or an alternative whose accuracy can be substantianted) in order for the entire investigation to be considered legitimate, if the basis of the physical argument (in colloids, biology, or any other application) depends on quantitative accuracy of particle positions. We compare our algorithms to other recent and related advances in location/tracking in colloids and in biology, and discuss the relative strengths and weaknesses of all the algorithms in various situations. We carry out performance tests directly comparing the accuracy of our and other 3D methods with simulated data for both location and tracking, and in providing relative performance data, we assess just how accurately software can locate particles. We discuss how our methods, now applied to colloids, could improve the location and tracking of features such as quantum dots in cells.

Dynamics of weakly aggregated colloidal particles

We discuss the behaviour of the dynamics of colloidal particles with a weak attractive interparticle interaction that is induced through the addition of polymer to the solvent. We briefly review the description of their behaviour in terms of the jamming phase diagram, which parametrized the fluid–to–solid transition due to changes in volume fraction, attractive energy or applied stress. We focus on a discussion of ageing of the solid gels formed by these colloid–polymer mixtures. They exhibit a delayed collapse induced by gravity. The time evolution of the height of the sediment exhibits an unexpected scaling behaviour, suggesting a universal nature to this delayed collapse. We complement these measurements of the scaling of the collapse with microscopic investigations of the evolution of the structure of the network using confocal microscopy. These results provide new insight into the origin of this ageing behaviour.

Stochastic variation: from single cells to superorganisms.

Observed phenotype often fails to correspond with genotype. Although it is well established that uncontrolled genetic modifier effects and environmental variability can affect phenotype, stochastic variation in gene expression can also contribute to phenotypic differences. Here we examine recent work that has provided insights into how fundamental physical properties of living cells, and the probabilistic nature of the chemical reactions that underlie gene expression, introduce noise. We focus on instances in which a stochastic decision initiates an event in the development of a multicellular organism and how that decision can be subsequently fixed. We present an example indicating that a similar interplay between an initial stochastic decision and subsequent fixation may underlie the regulation of reproduction in social insects. We argue, therefore, that stochasticity affects biological processes from the single‐gene scale through to the complex organization of an ant colony, and represents a largely neglected component of phenotypic variation and evolution.

A random walk description of the heterogeneous glassy dynamics of attracting colloids

We study the heterogeneous dynamics of attractive colloidal particles close to the gel transition using confocal microscopy experiments combined with a theoretical statistical analysis. We focus on single particle dynamics and show that the self-part of the van Hove distribution function is not the Gaussian expected for a Fickian process, but that it reflects instead the existence, at any given time, of colloids with widely different mobilities. Our confocal microscopy measurements can be described well by a simple analytical model based on a conventional continuous time random walk picture, as already found for several other glassy materials. In particular, the theory successfully accounts for the presence of broad tails in the van Hove distributions that exhibit exponential, rather than Gaussian, decay at large distance.

Universal behaviour in the mechanical properties of weakly aggregated colloidal particles

We study the evolution of the elastic shear modulus of weakly aggregated colloidal particles during the onset of a delayed collapse under gravity. The early lifetime is characterised by an elastic shear modulus that increases logarithmically in time, following which the gels experience a catastrophic failure and the elastic modulus drops dramatically. As the gel collapses, various complex behaviours are seen, including a temporary stabilisation against collapse, and reformation of a new gel with its own elastic modulus that follows its own trajectory to collapse. Time-lapsed images acquired of identical samples in a transparent cell used to calibrate the measured shear modulus values allow observation of the sample-spanning collective rearrangement involved in the collapse. The loss of propagation of elastic stress in the gel is observed to precede the bulk collapse in all samples, with the two events always well-separated in time. The evolution of the viscoelastic response across a range of colloid volume fractions and polymer concentrations is significantly simplified by scaling the rheology curves for each sample together. From the scaling, we show a critical onset of the elastic modulus as a function of these system parameters. Moreover, our analyses of the time scale for collapse of the elastic shear modulus and of the gel itself over a range of colloid volume fractions and the polymer concentrations support a simple phenomenological model based on the dependence of the microscopic dynamics on the strength and number of sticky interparticle bonds.

Experimental determination of order in non-equilibrium solids using colloidal gels

The idea of quantifying order in disordered systems has been introduced recently by Torquato and co-workers (2000 Phys. Rev. E 62 993–1001). We are interested in the application of this idea to measure structure in non-equilibrium systems. Here we focus on gels, using as a model system colloidal gels formed from hard spheres with polymer added to the systems to induce a controlled, weak attraction. To describe the structure of the gels we use real space imaging via confocal microscopy to obtain the full three-dimensional structure. We measure experimentally both translational order and bond angle correlations, defining a new (refined) translational order parameter that is sensitive to long range order in these non-random packings. This metric is also sensitive to anisotropy, which should be important in the many physical situations where an external force is present. The bond angle distribution shows coordinated organization. To give a clearer physical picture for gels, we compare the experimental data to computer generated hard sphere systems.

Imaging cellulose synthase motility during primary cell wall synthesis in the grass Brachypodium distachyon

The mechanism of cellulose synthesis has been studied by characterizing the motility of cellulose synthase complexes tagged with a fluorescent protein; however, this approach has been used exclusively on the hypocotyl of Arabidopsis thaliana. Here we characterize cellulose synthase motility in the model grass, Brachypodium distachyon. We generated lines in which mEGFP is fused N-terminal to BdCESA3 or BdCESA6 and which grew indistinguishably from the wild type (Bd21-3) and had dense fluorescent puncta at or near the plasma membrane. Measured with a particle tracking algorithm, the average speed of GFP-BdCESA3 particles in the mesocotyl was 164 ± 78 nm min−1 (error gives standard deviation [SD], n = 1451 particles). Mean speed in the root appeared similar. For comparison, average speed in the A. thaliana hypocotyl expressing GFP-AtCESA6 was 184 ± 86 nm min−1 (n = 2755). For B. distachyon, we quantified root diameter and elongation rate in response to inhibitors of cellulose (dichlorobenylnitrile; DCB), microtubules (oryzalin), or actin (latrunculin B). Neither oryzalin nor latrunculin affected the speed of CESA complexes; whereas, DCB reduced average speed by about 50% in B. distachyon and by about 35% in A. thaliana. Evidently, between these species, CESA motility is well conserved.

Ligand‐directed delivery of fluorophores to track native calcium‐permeable AMPA receptors in neuronal cultures

Subcellular trafficking of neuronal receptors is known to play a key role in synaptic development, homeostasis, and plasticity. We have developed a ligand‐targeted and photo‐cleavable probe for delivering a synthetic fluorophore to AMPA receptors natively expressed in neurons. After a receptor is bound to the ligand portion of the probe molecule, a proteinaceous nucleophile reacts with an electrophile on the probe, covalently bonding the two species. The ligand may then be removed by photolysis, returning the receptor to its non‐liganded state while leaving intact the new covalent bond between the receptor and the fluorophore. This strategy was used to label polyamine‐sensitive receptors, including calcium‐permeable AMPA receptors, in live hippocampal neurons from rats. Here, we describe experiments where we examined specificity, competition, and concentration on labeling efficacy as well as quantified receptor trafficking. Pharmacological competition during the labeling step with either a competitive or non‐competitive glutamate receptor antagonist prevented the majority of labeling observed without a blocker. In other experiments, labeled receptors were observed to alter their locations and we were able to track and quantify their movements. We used a small molecule, ligand‐directed probe to deliver synthetic fluorophores to endogenously expressed glutamate receptors for the purpose of tracking these receptors on live, hippocampal neurons. We found that clusters of receptors appear to move at similar rates to previous studies. We also found that the polyamine toxin pharmacophore likely binds to receptors in addition to calcium‐permeable AMPA receptors.

Self-Healing Biomaterials: Entangled DNA Networks

The material that the cell uses to store genetic information actually becomes self-healing material when it must be segregated, transported across the length of the cell even in the face of its entanglements with itself, or condensed to prepare for this large scale transport. This is a naturally-occuring soft material that is capable of self-repair. Moreover, there is evidence that the material first senses the possibility of a stress-inducing, potentially fatal (to genome stability) topological entanglement, and quickly fortifies itself to limit or prevent the damage. The issue of how topology is conserved (controlled) by this type of biomaterial is itself an interesting mystery. I will present our experimental results using a bottom-up design approach, borrowing the naturally-occurring materials from the cell to study the design principles of this behavior. We incorporated micron-sized particles in lambda-DNA entangled networks in the presence of the topoisomerase II motor that performs the strand passage, at controllable ratio of enzyme units per average DNA entanglement and ATP concentration. We used bright-field microscopy to directly track the movement of the particles, which couple to the DNA fluctuating movement. Our observed scaling behavior suggests entangled dynamics in the bare DNA system, and nonentangled Rouse dynamics, with enzyme performing topological constraint relaxation, in the DNA+topo II + ATP system. These very time-dependent scaling behaviors are all predicted theoretically for entangled polymers with inclusion of a constrained release process in the case of presence of active topo II. The material self-heals to such a degree that, at saturating topoisomerase II motor and ATP concentrations, the long DNA polymer molecules do not even “feel” one another despite being entangled. We compare our experimental results to predictions of the constraint release model, measuring the “healing rate” at different dynamical length scales.

Nonequilibrium Mechanics of the Mitotic Spindle Measured in Living Cells

To carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex non-equilibrium processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly of and maintenance of tension across the mitotic spindle, a nonequilibrium composite material including polymers and motor proteins that is responsible for organizing and separating the genetic material during cell division. The intrinsic microtubule dynamics and different motor proteins provide the forcing required for this dynamic process. We use high-resolution fluorescence confocal microscopy to observe and analyze the real space dynamics of the mitotic spindle in budding yeast and Drosophila melanogaster S2 cells. Centrosome trajectories are reconstructed from the three-dimensional fluorescence data and quantified in a coordinate system relevant to the cell division using specially-developed image analysis methods. The roles of specific motor proteins are isolated by altering their functionality through genetic and chemical means using a microfluidic device. We use the fluctuations in pre-anaphase centrosome positions to show how nonequilibrium motor activity controls the mechanical properties of an in vivo cytoskeletal network.View Large Image | View Hi-Res Image | Download PowerPoint Slide