Multiple Particle Tracking Research Articles

Micro-heterogeneity of cellulosic fiber biopolymer prepared from corn hulls

Z-trim is a zero calorie cellulosic fiber biopolymer produced from corn hulls. The micro-structural heterogeneities of Z-trim biopolymer were investigated by monitoring the thermally driven displacements of well-dispersed micro-spheres via video fluorescence microscopy named multiple-particle tracking (MPT). By comparing the distribution of the time-dependent mean-square displacement (MSD) of polystyrene micro-spheres embedded in three concentrations of Z-trim, we found that the degree of heterogeneity of the suspensions increased dramatically within a narrow range of concentrations. The ensemble-averaged MSD of 0.5 g/100 g Z-trim biopolymer exhibited a power-law behavior that scaled log-linearly with time lag. This behavior was similar to that of homogeneous aqueous glycerol solution. But the MSD distribution for Z-trim was wider and more asymmetric than for glycerol. Increasing Z-trim concentration caused more deviation of micro-spheres colloidal dispersion from homogeneity as detected by the MPT.

Probing the Micrheology of Mesenchymal Stem Cell Migration to Tumors

Mesenchymal stem cells (MSCs) are excellent candidates for the development of cell-based gene delivery systems; however, extended cell culture, required for therapeutic development, alters MSC morphology, reducing MSC migration upon reinfusion. Spontaneous migration of MSCs to tumors is mediated by tumor secretion of proangiogenic chemokines. Multiple particle tracking microrheology was used to investigate the effect of tumor-secreted molecules on MSC viscoelasticity, which was correlated with MSC migration and morphology. Within 24 hours after MSC treatment with tumor-conditioned media (TCM), MSCs were elongated, with more than 5-fold difference in the length of lamellipodia. Within 24 hours, the migration of MSCs, measured using a Boyden chamber assay, toward TCM was increased 10-fold over control media. The mean squared displacements (MSDs) of 100-nm carboxylated polystyrene particles, injected into the cytoplasm of human MSCs using the Biolistic Particle Injection System, were determined with 33 ms temporal and 5 nm spatial resolution using multiple particle tracking. The frequency dependent elastic and viscous moduli were calculated from the complex shear moduli, which were determined from the Fourier transform of the time-dependent MSDs, using the frequency-dependent Stokes-Einstein equation. Pretreatment of MSCs with TCM resulted in rapid changes in cytoplasmic viscoelasticity with a 9.8-fold increase in the average elastic moduli, which increased from 35 to 344 dyn/cm2, and a 3.5-fold decrease in the average viscous moduli, which was reduced from 99 to 28 dyn/cm2, within 1 hour (n = 6-8 cells per group). We hypothesize that tumor-secreted molecules increase MSC mobility by altering cytoskeletal organization. Changes in MSC viscosity may be in part to reduced actin cross-linking during cytoskeletal reorganization. Increased MSC rigidity may be due to MSC elongation, which leads to the formation of polymer entanglements as the ratio of cell length to width is greatly increased.

On the origins of the universal dynamics of endogenous granules in mammalian cells.

Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics--both of which are crucial for the interpretation of the results from MPTM technique--are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms--molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.

Combined Passive and Active Microrheology Study of Protein-Layer Formation at an Air−Water Interface

We investigate the mechanical properties of layers of the protein beta-lactoglobulin during their formation at the air-water interface using a combination of passive and active microrheological techniques. The passive microrheology, which employs multiple particle tracking measurements using spherical colloids, indicates that the interfacial rheology evolves over time through three stages as protein adsorbs at the interface: (i) an increase in viscosity, (ii) a period of spatial heterogeneity in which the interface contains elastic and viscous regions, and (iii) the development of a uniformly rigid elastic film. Varying solution pH between pH = 5.2, the isoelectric point of beta-lactoglobulin, and pH = 7.0 has no qualitative effect on this mechanical evolution. The active microrheology, which employs ferromagnetic nanowires rotating in response to magnetic torques, similarly shows an increasing interfacial viscosity at early times and evidence of mechanical heterogeneity at intermediate times. However, at late times, the nanowire mobility becomes strongly pH dependent. For pH = 5.2, the layer responds as a rigid elastic film to the stress imposed by the wire. For pH = 7.0, it displays a viscous response that contrasts with the passive measurements. We associate this contrast with a nonlinear response to the wire at late times that reflects a low yield stress of the film at higher pH. This ability to compare passive and active measurements demonstrates the advantage of applying multiple microrheological methods to resolve ambiguity in any single approach.

Sequence-dependent gelation kinetics of β-hairpin peptide hydrogels.

The gelation kinetics of four β-hairpin oligopeptides that have been designed to exhibit responsive behavior to changes in environmental conditions, such as pH, ionic strength and temperature, are characterized using multiple particle tracking microrheology and circular dichroism (CD) spectroscopy. The peptides, predominantly an alternating sequence of valine and lysine residues, differ by a point substitution of a single amino acid near a type II'β-turn sequence. The rate of gelation becomes faster for point substitutions which reduce the total charge of the peptide. Similarly, increasing the ionic strength reduces or screens intra- and inter-molecular electrostatic repulsions, again leading to faster gelation kinetics. CD measurements show that the concentration of folded peptide at the gel point decreases as the gelation kinetics become slower, possibly indicating a relationship between the assembly rate and the resulting gel microstructure. Finally, a model is developed based on the electrostatic barrier to peptide folding and association which agrees semi-quantitatively with the microrheology results. This represents a first step towards understanding the role of peptide charge and physico-chemical conditions in the self-assembly of these peptide hydrogelators.

Mechanical changes of living oocytes at maturation investigated by multiple particle tracking

We have studied the mechanical properties of the cytoplasm of starfish oocytes before and after maturation using the multiple particle tracking technique. Fluorescent microbeads are microinjected in oocytes of Astropecten aranciacus starfishes. Mechanical properties are obtained analyzing the mean-square displacement (MSD) of the beads trajectories. The ensemble-averaged MSD provides information about diffusion coefficient D, and the cytoplasm viscosity η is estimated through the Stokes–Einstein equation. Results show that maturation is correlated with change of cytoplasm viscosity. The results can be ascribed to F-actin reorganization as also supported by fluorescence confocal microscopy.

Multiple Particle Tracking Investigations of Acid Milk Gels Using Tracer Particles with Designed Surface Chemistries and Comparison with Diffusing Wave Spectroscopy Studies

Multiple particle tracking (MPT) has been used in an attempt to probe the heterogeneity of acid milk gels, made with and without added pectin, by following the distribution of the displacements of added tracer beads during and after gelation using the Van Hove distribution. Furthermore, the surface chemistry of the latex probe particles was modified in an attempt to control their location in the system and probe the microrheological properties of the protein network and aqueous-phase voids independently. In addition, the mean square displacement (MSD) of the casein micelles/casein aggregates themselves, obtained by diffusing wave spectroscopy (DWS), has been compared to the ensemble-averaged MSD calculated from the data obtained by tracking the movement of the added tracers, with and without a kappa-casein coating. For the kappa-casein-coated tracer particles, upon acidification and subsequent gel formation, the MSDs obtained by MPT superimpose remarkably well with the MSDs obtained by DWS, despite the fact that one is obtained by tracking the movement of the particle network elements themselves and the other is obtained from directly tracking added tracers. This result has important implications: (i) it demonstrates that, although the DWS measurement is intrinsically ensemble-averaged, it really gives insight into the dynamics of the colloidal gel network; (ii) it confirms that the kappa-casein-coated probes used in this MPT experiment are well incorporated into the gel network; and hence (iii) that at least in gelled systems kappa-casein-coated latex probes are interesting probes that reveal the dynamics of the casein network.

Gelation of Covalently Cross-Linked PEG−Heparin Hydrogels

We study PEG-heparin hydrogels to identify compositions that lead to gel formation and measure the corresponding gelation kinetics. The material consists of a maleimide-functionalized high molecular weight heparin (HMWH) backbone covalently cross-linked with bis-thiol poly(ethylene glycol) (PEG). Using multiple particle tracking microrheology, we investigate a broad composition space, defined by the number of maleimide functional sites per HMWH (f = 3.9-11.8), the molecular weight of the PEG cross-linker (M(n) = 2000, 5000, and 10 000), and the concentrations of the heparin and PEG polymers. Gelation kinetics are characterized by time-cure superposition, yielding the gel time, t(c), and the critical relaxation exponent, n. Gelation times range from 5 < t(c) ≤ 45 min, with the fastest kinetics occurring for the highest HMWH maleimide functionalities. t(c) depends nonmonotonically on the PEG cross-linker molecular weight, suggesting that gelation is affected by the length of the cross-linker relative to intermolecular interactions between heparin molecules. The critical relaxation exponent decreases from n = 0.52 for PEG 2000 to n = 0.39 for PEG 10 000. Finally, 219 equilibrated samples taken over the entire composition space are identified as liquid or solid, defining the "gelation envelope". The boundaries of this empirical gelation envelope are in good agreement with Flory-Stockmayer theory. In all, microrheological measurements enable characterization over a large parameter space and provide crucial insight into the gelation of complex, multifunctional hydrogelators used in therapeutic applications.

Mapping Local Matrix Remodeling Induced by a Migrating Tumor Cell Using Three-Dimensional Multiple-Particle Tracking

Mesenchymal cell migration through a three-dimensional (3D) matrix typically involves major matrix remodeling. The direction of matrix deformation occurs locally in all three dimensions, which cannot be measured by current techniques. To probe the local, 3D, real-time deformation of a collagen matrix during tumor cell migration, we developed an assay whereby matrix-embedded beads are tracked simultaneously in all three directions with high resolution. To establish a proof of principle, we investigated patterns of collagen I matrix deformation near fibrosarcoma cells in the absence and presence of inhibitors of matrix metalloproteinases and acto-myosin contractility. Our results indicate that migrating cells show patterns of local matrix deformation toward the cell that are symmetric in magnitude with respect to the axis of cell movement. In contrast, patterns of matrix release from the cell are asymmetric: the matrix is typically relaxed first at the back of the cell, allowing forward motion, and then at the cell's leading edge. Matrix deformation in regions of the matrix near the cell's leading edge is elastic and mostly reversible, but induces irreversible matrix rupture events near the trailing edge. Our results also indicate that matrix remodeling spatially correlates with protrusive activity. This correlation is mediated by myosin II and Rac1, and eliminated after inhibition of pericellular proteolysis or ROCK. We have developed an assay based on high-resolution 3D multiple-particle tracking that allows us to probe local matrix remodeling during mesenchymal cell migration through a 3D matrix and simultaneously monitor protrusion dynamics.

Microrheological studies reveal semiflexible networks in gels of a ubiquitous cell wall polysaccharide

Microrheological measurements have been carried out on ionotropic gels made from an important cell wall polysaccharide, using diffusing wave spectroscopy and multiple particle tracking. These gels were formed by the interaction of calcium ions with negatively charged groups on the polymer backbone, which is a copolymer of charged and uncharged sugars, galacturonic acid, and its methylesterified analog, respectively. The results suggest that semiflexible networks are formed in these systems, with a low frequency, frequency independent storage modulus (G'>G''), and a high frequency scaling of both G' and G'' with omega3/4. The differences observed between gels obtained using polysaccharide samples with different amounts and patterns of the charged ion-binding groups could comfortably be accommodated within this theoretical framework, assuming that the elementary semiflexible elements of the network are filaments consisting of two polymer chains bridged with calcium. In particular, a sample that was engineered to possess a blockwise intramolecular distribution of calcium chelating moieties clearly exhibited the high frequency scaling of both moduli with omega3/4 across some three orders of magnitude, and the concentration dependences of the elastic modulus, at both high and low frequency, were found to follow power laws with predicted exponents. Furthermore, quantitative agreement of the moduli with theory was found for realistic estimates of the molecular parameters, suggesting that the physics of semiflexible networks is not only exploited by protein components of the cytoskeleton but also by polysaccharides in plant cell walls.

Statistical and sampling issues when using multiple particle tracking

Video microscopy can be used to simultaneously track several microparticles embedded in a complex material. The trajectories are used to extract a sample of displacements at random locations in the material. From this sample, averaged quantities characterizing the dynamics of the probes are calculated to evaluate structural and/or mechanical properties of the assessed material. However, the sampling of measured displacements in heterogeneous systems is singular because the volume of observation with video microscopy is finite. By carefully characterizing the sampling design in the experimental output of the multiple particle tracking technique, we derive estimators for the mean and variance of the probes' dynamics that are independent of the peculiar statistical characteristics. We expose stringent tests of these estimators using simulated and experimental complex systems with a known heterogeneous structure. Up to a certain fundamental limitation, which we characterize through a material degree of sampling by the embedded probe tracking, these estimators can be applied to quantify the heterogeneity of a material, providing an original and intelligible kind of information on complex fluid properties. More generally, we show that the precise assessment of the statistics in the multiple particle tracking output sample of observations is essential in order to provide accurate unbiased measurements.

Microrheological studies offer insights into polysaccharide gels

Microrheological measurements have been carried out on a series of model systems of increasing structural and temporal complexity using multiple particle tracking (MPT). Purely viscous media, entangled polymeric solutions, and subsequently a biologically relevant polysaccharide gel have all been studied. In addition, the gelled sample has been mechanically disrupted in order to induce a transient heterogeneous microstructure. For the polysaccharide sample MPT results are analysed in order to yield both one- and two-point microrheological measurements and these are compared with those obtained by additional experiments carried out using diffusing wave spectroscopy (DWS) and those obtained using a conventional rheometer. Results from glycerol/water mixtures and polyethylene oxide solutions agree with expectations, providing confidence both in the techniques used and the analysis employed. Moreover, a calcium-induced gel of the polysaccharide pectin yields similar results to those published recently on actin, with the elastic modulus being frequency independent at low frequency, and both elastic and viscous moduli exhibiting a high frequency scaling with ω 3/4. Furthermore, preliminary results reported here indicate that these techniques are ideally suited to the study of the temporal evolution of the spatial distribution of mechanical properties in such samples, for example during gelation or digestion.

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Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus.

Nanoparticles larger than the reported mesh-pore size range (10-200 nm) in mucus have been thought to be much too large to undergo rapid diffusional transport through mucus barriers. However, large nanoparticles are preferred for higher drug encapsulation efficiency and the ability to provide sustained delivery of a wider array of drugs. We used high-speed multiple-particle tracking to quantify transport rates of individual polymeric particles of various sizes and surface chemistries in samples of fresh human cervicovaginal mucus. Both the mucin concentration and viscoelastic properties of these cervicovaginal samples are similar to those in many other human mucus secretions. Unexpectedly, we found that large nanoparticles, 500 and 200 nm in diameter, if coated with polyethylene glycol, diffused through mucus with an effective diffusion coefficient (D(eff)) only 4- and 6-fold lower than that for the same particles in water (at time scale tau = 1 s). In contrast, for smaller but otherwise identical 100-nm coated particles, D(eff) was 200-fold lower in mucus than in water. For uncoated particles 100-500 nm in diameter, D(eff) was 2,400- to 40,000-fold lower in mucus than in water. Much larger fractions of the 100-nm particles were immobilized or otherwise hindered by mucus than the large 200- to 500-nm particles. Thus, in contrast to the prevailing belief, these results demonstrate that large nanoparticles, if properly coated, can rapidly penetrate physiological human mucus, and they offer the prospect that large nanoparticles can be used for mucosal drug delivery.

Electrostatically tuned rate of peptide self-assembly resolved by multiple particle tracking

Hydrogels formed from the self-assembly of oligopeptides are being extensively studied for biomedical applications. The kinetics of their gelation, as well as a quantitative description of the forces controlling the rate of assembly has not yet been addressed. We report here the use of multiple particle tracking to measure the self-assembly kinetics of the model peptide FKFEFKFE (KFE8). KFE8 forms well-defined β-sheet intermediates and is often used as a model peptide system that forms a fibrous network in aqueous solvent. We find that increasing the pH of this system from 3.5 to 4.0 decreases the time of KFE8 gelation by almost hundredfold, from hours to minutes. A remarkable self-similarity between measurements performed at different pH suggests that, although accelerated by the pH increase, gelation follows an invariable mechanism. We propose a semi-quantitative interpretation for the order of magnitudes of gelation time using a simple model for the interaction driving the self-assembly in terms of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Such understanding is important for the development of current and future therapeutic applications ( drug delivery).

Microrheology and ROCK Signaling of Human Endothelial Cells Embedded in a 3D Matrix

Cell function is profoundly affected by the geometry of the extracellular environment confining the cell. Whether and how cells plated on a two-dimensional matrix or embedded in a three-dimensional (3D) matrix mechanically sense the dimensionality of their environment is mostly unknown, partly because individual cells in an extended matrix are inaccessible to conventional cell-mechanics probes. Here we develop a functional assay based on multiple particle tracking microrheology coupled with ballistic injection of nanoparticles to measure the local intracellular micromechanical properties of individual cells embedded inside a matrix. With our novel assay, we probe the mechanical properties of the cytoplasm of individual human umbilical vein endothelial cells (HUVECs) embedded in a 3D peptide hydrogel in the presence or absence of vascular endothelial growth factor (VEGF). We found that VEGF treatment, which enhances endothelial migration, increases the compliance and reduces the elasticity of the cytoplasm of HUVECs in a matrix. This VEGF-induced softening response of the cytoplasm is abrogated by specific Rho-kinase (ROCK) inhibition. These results establish combined particle-tracking microrheology and ballistic injection as the first method able to probe the micromechanical properties and mechanical response to agonists and/or drug treatments of individual cells inside a matrix. These results suggest that ROCK plays an essential role in the regulation of the intracellular mechanical response to VEGF of endothelial cells in a 3D matrix.

A Network-based Analysis of Polyanion-binding Proteins Utilizing Yeast Protein Arrays

The high affinity of certain cellular polyanions for many proteins (polyanion-binding proteins (PABPs)) has been demonstrated previously. It has been hypothesized that such polyanions may be involved in protein structure stabilization, stimulation of folding through chaperone-like activity, and intra- and extracellular protein transport as well as intracellular organization. The purpose of the proteomics studies reported here was to seek evidence for the idea that the nonspecific but high affinity interactions of PABPs with polyanions have a functional role in intracellular processes. Utilizing yeast protein arrays and five biotinylated cellular polyanion probes (actin, tubulin, heparin, heparan sulfate, and DNA), we identified proteins that interact with these probes and analyzed their structural and amino acid sequence requirements as well as their predicted functions in the yeast proteome. We also provide evidence for the existence of a network-like system for PABPs and their potential roles as critical hubs in intracellular behavior. This investigation takes a first step toward achieving a better understanding of the nature of polyanion-protein interactions within cells and introduces an alternative way of thinking about intracellular organization.

Multiple-particle tracking—an improvement for positron particle tracking

The Birmingham positron camera for tracking positron-emitting particles has been successfully applied in a wide range of fundamental research and industrial applications. The technique was developed to track a single tracer; however, tracking multiple particles would provide more comparative information on the physical processes taking place in a system. Here, multiple-particle tracking, a new method that is able to track more than one particle, is presented. The locations of each tracer are calculated in three dimensions by taking account of the radioactivity concentration of each tracer and by discarding corrupted γ-ray trajectories. The potential applications of the multiple-particle tracking technique are illustrated through two examples by tracking three stationary and three moving particles, respectively.

Multiple particle tracking in 3-D+t microscopy: method and application to the tracking of endocytosed quantum dots

We propose a method to detect and track multiple moving biological spot-like particles showing different kinds of dynamics in image sequences acquired through multidimensional fluorescence microscopy. It enables the extraction and analysis of information such as number, position, speed, movement, and diffusion phases of, e.g., endosomal particles. The method consists of several stages. After a detection stage performed by a three-dimensional (3-D) undecimated wavelet transform, we compute, for each detected spot, several predictions of its future state in the next frame. This is accomplished thanks to an interacting multiple model (IMM) algorithm which includes several models corresponding to different biologically realistic movement types. Tracks are constructed, thereafter, by a data association algorithm based on the maximization of the likelihood of each IMM. The last stage consists of updating the IMM filters in order to compute final estimations for the present image and to improve predictions for the next image. The performances of the method are validated on synthetic image data and used to characterize the 3-D movement of endocytic vesicles containing quantum dots.

Intracellular Transport Dynamics of Endosomes Containing DNA Polyplexes along the Microtubule Network

We have explored the transport of DNA polyplexes enclosed in endosomes within the cellular environment by multiple particle tracking (MPT). The polyplex-loaded endosomes demonstrate enhanced diffusion at short timescales ( t < 7 s) with their mean-square displacement (MSD) 〈Δ x( t) 2 〉 scaling as t 1.25. For longer time intervals they exhibit subdiffusive transport and have an MSD scaling as t 0.7. This crossover from an enhanced diffusion to a subdiffusive regime can be explained by considering the action of motor proteins that actively transport these endosomes along the cellular microtubule network and the thermal bending modes of the microtubule network itself.