Thomas A. Waigh

Microrheology of complex fluids

The field of microrheology is concerned with how materials store and dissipate mechanical energy as a function of length scale. Recent developments in the theory and instrumentation of the microrheology of complex fluids are reviewed. Equal emphasis is given to the physical phenomena probed, advances in instrumentation, and specific experimental systems in which this field has already had an impact. The inversion of the compliance data, measurement of sample heterogeneity, high frequency viscoelasticity, effects of shear flow, single molecule experiments, surface viscoelasticity and time evolution studies are considered. The techniques highlighted include particle tracking microrheology, diffusing wave spectroscopy, laser tracking, magnetic tweezers and atomic force microscopy. Specific examples of complex fluid systems are chosen from the fields of polymers, colloids and biological assemblies.

The phase transformations in starch during gelatinisation: a liquid crystalline approach.

The analogy between starch and a chiral side-chain polymeric liquid crystal is examined in relation to the processes involved during gelatinisation. There are three important parameters for characterisation of the molecular phase behaviour of the amylopectin: the lamellar order parameter (psi), the orientational order parameter of the amylopectin double helices (phi), and the helicity of the sample (h, the helix/coil ratio, a measure of the helix-coil transition of the double helices). The coupling between the double helices and the backbone through the flexible spacers is affected dramatically by the water content and it is this factor which dictates the particular phase adopted by the amylopectin inside the starch granule as a function of temperature. SAXS, WAXS and 13C CP/MAS NMR are used to examine these phenomena in excess water. Furthermore, previous experimental evidence pertaining to the limiting water case is reviewed with respect to this new theoretical framework.

Scattering Studies of the Internal Structure of Starch Granules

Small Angle X-ray Scattering (SAXS) and Small Angle Neutron Scattering (SANS) are applied to the study of the internal supramolecular packing within starch granules. SAXS is able to provide information on the packing of the semicrystalline lamellae and the amorphous growth rings. Using synchrotron radiation it is possible to follow changes in structure at both elevated temperatures within the gelatinisation regime, and at sub-zero temperatures when ice crystallisation can change the packing via compression of the granule. The role of amylose in modifying the response of the granule to these temperature variations has been examined. SANS has been used to examine the distribution of water in different parts of the granule, and quantify the molecular densities in different regions of the granule.

Particle tracking microrheology of purified gastrointestinal mucins.

The rheological characteristics of gastric and duodenal mucin solutions, the building blocks of the mucus layer that covers the epithelia of the two organs, were investigated using particle tracking microrheology. We used biochemically well characterized purified porcine mucins (MUC5AC and MUC2) as models for human mucins, to probe their viscoelasticity as a function of mucin concentration and pH. Furthermore, we used both reducing (dithiothreitol, DTT) and chaotropic agents (guanidinium chloride and urea) to probe the mesoscopic forces that mediate the integrity of the polymer network. At neutral pH both gastric and duodenal mucins formed self-assembled semi-dilute networks above a certain critical mucin concentration (c*) with the viscosity (η) scaling as η∼c(0.53±0.08) for MUC5AC and η∼c(0.53±0.06) for MUC2, where c is the mucin concentration. Above an even higher mucin concentration threshold (ce , the entanglement concentration) reptation occurs and there is a dramatic increase in the viscosity scaling, η∼c(3.92±0.38) for MUC5AC and η∼c(5.1±0.8) for MUC2. The dynamics of the self-assembled comb polymers is examined in terms of a scaling model for flexible polyelectrolyte combs. Both duodenum and gastric mucin are found to be pH switchable gels, gelation occurring at low pHs. There is a hundred-fold increase in the elastic shear modulus once the pH is decreased. The addition of DTT, guanidinium chloride and urea disassembles both the semi-dilute and gel structures causing a large increase in the compliance (decrease in their shear moduli). Addition of the polyphenol EGCG has a reverse effect on mucin viscoelasticity, that is, it triggers a sol-gel transition in semi-dilute mucin solutions at neutral pH.

Intracellular microrheology of motile amoeba proteus

The motility of Amoeba proteus was examined using the technique of passive particle tracking microrheology, with the aid of newly developed particle tracking software, a fast digital camera, and an optical microscope. We tracked large numbers of endogeneous particles in the amoebae, which displayed subdiffusive motion at short timescales, corresponding to thermal motion in a viscoelastic medium, and superdiffusive motion at long timescales due to the convection of the cytoplasm. Subdiffusive motion was characterized by a rheological scaling exponent of 3/4 in the cortex, indicative of the semiflexible dynamics of the actin fibers. We observed shear-thinning in the flowing endoplasm, where exponents increased with increasing flow rate; i.e., the endoplasm became more fluid-like. The rheology of the cortex is found to be isotropic, reflecting an isotropic actin gel. A clear difference was seen between cortical and endoplasmic layers in terms of both viscoelasticity and flow velocity, where the profile of the latter is close to a Poiseuille flow for a Newtonian fluid.

Double-globular structure of porcine stomach mucin: a small-angle X-ray scattering study

We present evidence from small-angle X-ray scattering synchrotron experiments that porcine stomach mucin (MUC6) contains a double-globular comb structure. Analysis of the amino acid sequence of the peptide comb backbone indicates that the globular structure is determined by both the charge and hydrophobicity of the amino acids and the placement of the short hydrophilic carbohydrate side chains (approximately 2.5 nm). The double-globular structure is, thus, due to a block copolymer type hydrophobic polyampholyte charge instability in contrast to the random copolymer instabilities observed previously with synthetic polyelectrolytes (particularly polystyrene sulfonates). Careful filtering was required to exclude multimonomer aggregates from the X-ray measurements. A double Guinier analysis ( R g approximately 26 nm) and a double power law fit are consistent with two globules per chain in low salt conditions. The average radius of the globules is approximately 10 nm in salt- free condition (double Guinier fit) and the average distance of intrachain separation of the globules is 48 nm. The addition of salt causes a significant decrease in the radius of gyration (14 nm 100 mM NaCl) of the chains and is attributed to the contraction of the glycosylated peptide spacer between the two globules (the globular size continues to be approximately 10 nm and the globule separation is then 18 nm). Without salt, the scaling of the semidilute mesh size (xi) as a function of the mucin concentration (c) is xi approximately c (-0.45)compared with xi approximately c (-0.28) in high salt conditions, highlighting the globular nature of the chains. In contrast, hydrophilic flexible polyelectrolytes have a stronger concentration dependence of xi when excess salt is added.

Roles of Dynein and Dynactin in Early Endosome Dynamics Revealed Using Automated Tracking and Global Analysis

Microtubule-dependent movement is crucial for the spatial organization of endosomes in most eukaryotes, but as yet there has been no systematic analysis of how a particular microtubule motor contributes to early endosome dynamics. Here we tracked early endosomes labeled with GFP-Rab5 on the nanometer scale, and combined this with global, first passage probability (FPP) analysis to provide an unbiased description of how the minus-end microtubule motor, cytoplasmic dynein, supports endosome motility. Dynein contributes to short-range endosome movement, but in particular drives 85–98% of long, inward translocations. For these, it requires an intact dynactin complex to allow membrane-bound p150Glued to activate dynein, since p50 over-expression, which disrupts the dynactin complex, inhibits inward movement even though dynein and p150Glued remain membrane-bound. Long dynein-dependent movements occur via bursts at up to ∼8 µms−1 that are linked by changes in rate or pauses. These peak speeds during rapid inward endosome movement are still seen when cellular dynein levels are 50-fold reduced by RNAi knock-down of dynein heavy chain, while the number of movements is reduced 5-fold. Altogether, these findings identify how dynein helps define the dynamics of early endosomes.

Advances in the microrheology of complex fluids

New developments in the microrheology of complex fluids are considered. Firstly the requirements for a simple modern particle tracking microrheology experiment are introduced, the error analysis methods associated with it and the mathematical techniques required to calculate the linear viscoelasticity. Progress in microrheology instrumentation is then described with respect to detectors, light sources, colloidal probes, magnetic tweezers, optical tweezers, diffusing wave spectroscopy, optical coherence tomography, fluorescence correlation spectroscopy, elastic- and quasi-elastic scattering techniques, 3D tracking, single molecule methods, modern microscopy methods and microfluidics. New theoretical techniques are also reviewed such as Bayesian analysis, oversampling, inversion techniques, alternative statistical tools for tracks (angular correlations, first passage probabilities, the kurtosis, motor protein step segmentation etc), issues in micro/macro rheological agreement and two particle methodologies. Applications where microrheology has begun to make some impact are also considered including semi-flexible polymers, gels, microorganism biofilms, intracellular methods, high frequency viscoelasticity, comb polymers, active motile fluids, blood clots, colloids, granular materials, polymers, liquid crystals and foods. Two large emergent areas of microrheology, non-linear microrheology and surface microrheology are also discussed.

Interfacial dynamic adsorption and structure of molecular layers of peptide surfactants.

Short peptide surfactants have recently emerged as a new class of amphiphiles, with tremendous potential in improving surface biocompatibility and mediating interfacial DNA immobilization. To establish their basic interfacial adsorption properties, cationic peptide surfactants V(m)K(n) have been studied by combining the measurements of spectroscopic ellipsometry (SE), neutron reflection (NR) and atomic force microscopy (AFM). Our results showed that changes in peptide structure, concentration, solution pH and ionic strength all affected their interfacial assembly. Increases in m and decreases in n reduced the critical aggregation concentration (CAC), but increased the amount of adsorption, showing the strong influence of the amphiphilic balance between hydrophilic and hydrophobic moieties. While the surface adsorbed amount increased with time and peptide concentration, an increase in ionic strength decreased peptide adsorption due to surface charge neutralization. Changes in solution pH did not affect the equilibrium surface adsorbed amount on the weakly negative SiO(2) surface, but did alter the adsorption dynamics. Neutron reflection revealed that V(6)K readily formed a bilayer structure of 35 A thickness at the interface, with the main part of the V(6) fragments being packed back-to-back to form a 15 A hydrophobic core and the two outer K regions being incorporated with a minor amount of V fragments forming the headgroup layers of 9 A each. AFM imaging revealed a sheet-like membrane structure incorporating defects of holes but the thicknesses probed by AFM were consistent with neutron reflection. It was demonstrated that the V(6)K peptide bilayer was effective for immobilization of DNA. The amount of DNA immobilized followed approximate 1:1 charge neutralization between the outer leaf peptide sublayer and the negatively charged DNA.

Reorganisation of the salivary mucin network by dietary components: Insights from green tea polyphenols

The salivary mucins that include MUC5B (gel-forming) and MUC7 (non-gel-forming) are major contributors to the protective mucus barrier in the oral cavity, and it is possible that dietary components may influence barrier properties. We show how one dietary compound, the green tea polyphenol epigallocatechin gallate (EGCG), can substantially alter the properties of both the polymeric MUC5B network and monomeric MUC7. Using rate-zonal centrifugation, MUC5B in human whole saliva and MUC5B purified from saliva sedimented faster in the presence of EGCG. The faster sedimentation by EGCG was shown to be greater with increasing MUC5B concentration. Particle tracking microrheology was employed to determine the viscosity of purified MUC5B solutions and showed that for MUC5B solutions of 200–1600 µg/mL, EGCG caused a significant increase in mucin viscosity, which was greater at higher MUC5B concentrations. Visualisation of the changes to the MUC5B network by EGCG was performed using atomic force microscopy, which demonstrated increased aggregation of MUC5B in a heterogeneous manner by EGCG. Using trypsin-resistant, high-molecular weight oligosaccharide-rich regions of MUC5B and recombinant N-terminal and C-terminal MUC5B proteins, we showed that EGCG causes aggregation at the protein domains of MUC5B, but not at the oligosaccharide-rich regions of the mucin. We also demonstrated that EGCG caused the majority of MUC7 in human whole saliva to aggregate. Furthermore, purified MUC7 also underwent a large increase in sedimentation rate in the presence of EGCG. In contrast, the green tea polyphenol epicatechin caused no change in the sedimentation rate of either MUC5B or MUC7 in human whole saliva. These findings have demonstrated how the properties of the mucin barrier can be influenced by dietary components. In the case of EGCG, these interactions may alter the function of MUC5B as a lubricant, contributing to the astringency (dry puckering sensation) of green tea.