No description available.

This diploma thesis deals with microrheology measured via the fluorescence correlation spectroscopy. As microrheological probes, fluorescently marked nanoparticles of 5 sizes in the range of 10-100 nm, were used. The particles had been immersed in a variety of concentrated glycerol solutions and agarose gels of different concentrations, and the FCS measurement revealed a diffusion coefficient of individual particles in each environment. Based on the coefficient, the viscosity of the glycerol needed to stop the particles could be determined. Particles of 10 nm size were not stopped even by the 100 wt% glycerol. In the case of the agarose gels, a combination of higher agarose concentration and larger particles resulted in an increase in the diffusion coefficient to an unlikely high value. This was caused probably by an agarose autofluorescence and the value indicates stopping of the particles in the given agarose gel. Later, the data acquired by the FCS measurement were converted to MSD curves using MATLAB software. The thesis discusses the influence of the experimental parameters on the shape of the MSD curve. The results showed that the number of particles and autocorrelation function have the most significant effect.

Adapting the tools of optical microscopy to the large-scale dynamic systems encountered in the development of multicellular organisms provides a path toward understanding the physical processes necessary for complex life to form and function. Obtaining quantitatively meaningful results from such systems has been challenging due to difficulty spanning the spatial and temporal scales representative of the whole, while also observing the many individual members from which complex and collective behavior emerges. A three-dimensional imaging technique known as light sheet fluorescence microscopy provides a number of significant benefits for surmounting these challenges and studying developmental systems. A thin plane of fluorescence excitation light is produced such that it coincides with the focal plane of an imaging system, providing rapid acquisition of optically sectioned images that can be used to construct a three-dimensional rendition of a sample. I discuss the implementation of this technique for use in larva of the model vertebrate Danio rerio (zebrafish). The nature of light sheet imaging makes it especially well suited to the study of large systems while maintaining good spatial resolution and minimizing damage to the specimen from excessive exposure to excitation light. I show the results from a comparative study that demonstrates the ability to image certain developmental processes non-destructively, while in contrast confocal microscopy results in abnormal growth due to phototoxicity. I develop the application of light sheet microscopy to the study of a previously inaccessible system: the bacterial colonization of a host organism. Using the technique, we are able to obtain a survey of the intestinal tract of a larval zebrafish and observe the location of microbes as they grow and establish a stable population in an initially germ free fish. Finally, I describe a new technique to measure the fluid viscosity of this intestinal environment in vivo using magnetically driven particles. By imaging such particles as they are oscillated in a frequency chirped field, it is possible to calculate properties such as the viscosity of the material in which they are embedded. Here I provide the first known measurement of intestinal mucus rheology in vivo. This dissertation includes previously published co-authored material.

DNA scaffolds self-assembled by short-stranded synthetic DNA can be tailored to build thermally reversible hydrogels with target binding sites. These hydrogels exhibit highly selective binding properties due to the specificity of DNA and also provide an aqueous environment for various reactions to happen within the network constraints. Hence, a careful study on the assembly mechanism and other physical aspects of DNA hydrogels is required to facilitate the future design and construction of such materials at the precise control. In this thesis, I present the work on well-designed DNA nano-stars as scaffolds for functional bulk materials with potential applications in bio-sensing. Chapter 1 starts with introducing the fundamental properties of DNA molecules, focusing on the advantages of utilising short-stranded DNA to programme and engineer micro- and macro- materials. Then it briefly reviews the field of rheology and micro-rheology, with the diffusing wave spectroscopy (DWS) technique illustrated explicitly as an example passive micro-rheology tool. Afterwards, a critical literature review on computational modelling of DNA systems is present, followed by the thesis outline at the end. Chapter 2 describes a simple DNA dendrimer system self-assembled from three-armed DNA nano-stars. The characterisation tools such as UV-vis spectroscopy, gel electrophoresis and dynamic light scattering (DLS) are introduced to verify the final production of the complex DNA structures. From this practice, we develop a routine for designing DNA scaffolds that yield optimal productivity. Chapter 3 investigates the mechanical properties of DNA hydrogels made of three-armed DNA nano-stars and how they change upon cooling and heating empolying DWS micro-rheology. The resulting viscoelastic moduli over a broad range of frequencies reveal a clear, temperature-reversible percolation transition coinciding with the melting temperature of the system's sticky ends. This indicates that we can achieve precise control in mechanical properties of DNA hydrogels, which is beneficial for designing more sensitive molecular sensing tools and controlled release systems. Chapter 4 develops a coarse-graining computational model of DNA hydrogels that resembles the system in Chapter 3 using LAMMPS, a classical molecular dynamics code. Thermodynamics, structural analysis and rheology tests were taken, qualitatively reproducing the physical phenomena of DNA assembly of the hydrogel network. Chapter 5 studies the internal behaviours of three-armed DNA complexes using oxDNA model also implemented in LAMMPS, with particular focus on the effect of the inert bases in the core and between double-stranded branches and single-stranded sticky ends. A deep insight into sequence-dependent behaviour of such complex structures can guide the parameter optimisation of the individual building blocks for the model described in Chapter 4. Chapter 6 concludes the thesis and presents an outlook for the future work that emerged out of my experimental and numerical studies.

The broad objective of my research is to investigate the physical characteristics and interactions of macromolecules and nanoparticles, and the corresponding effects on their phase separation behavior using static and dynamic light scattering (SLS & DLS). Light scattering provides a non-invasive technique for monitoring the in-situ behavior of solutes in solution, including solute interactions, sizes, shapes, aggregation kinetics and even rheological properties of condensed phases. Initially, we investigated lysozyme solutions for the presence of preformed aggregates and clusters that can distort the kinetics of protein crystal nucleation studies in this important model system for protein crystallization. We found that both undersaturated and supersaturated lysozyme solutions contained population of large, pre-existing protein aggregate. Separating these clusters and analyzing their composition with gel chromatography indicated that these clusters represented pre-formed lysozyme aggregates, and not extrinsic protein contamination. We investigated the effect of chaotropic versus kosmotropic ions (water structure breakers vs. structure makers) on the hydration layer and hydrodynamic interactions of hen egg white lysozyme. Surprisingly, neither chaotropic nor kosmotropic ions affected the protein hydration layer. Salt-effects on direct and hydrodynamic protein interactions were determined as function of the solutions ionic strength and temperature. Using both static and dynamic light scattering, we investigated the nucleation of gold nanoparticles forming from supersaturated gold sols. We observed that two well separated populations of nuclei formed essentially simultaneously, with sizes of 3nm vs. several tens of nanometer, respectively. We explore the use of lysozyme as tracer particle for diffusion-base measurements of electrolyte solutions. We showed that the unusual stability of lysozyme and its enhanced colloidal stability enable viscosity measurement of salts solutions at high salt concentration, over a wide range of pH values and temperatures for the common tracer particle polystyrene flocculates. We applied dynamic light scattering to measure the viscoelastic responses of polystyrene probe particles embedded in solutions and gels of two different polymers: polyacrylamide (PAAm) and poly-N-isopropylacrylamide (poly-NiPAAm).

The fundamentals of the photopolymerization process are not well understood. As a result, issues affecting the cure speed and overall quality of the final product (shape, size, and surface finish) of photopolymerization impose significant limitations on applications that require fast processing and high spatial resolution. To address this issue, microrheology was employed to perform in-situ monitoring of the liquid-to-gel transition during free-radical photopolymerization. Photosensitive acrylate and hydrogel resins were exposed to ultraviolet light, while the Brownian motion of micrometer sized, inert fluorescent tracer particles was tracked via optical videomicroscopy. Statistical analysis of particle motion yielded the rheological properties of the embedding medium as a function of time and location, thereby relating UV exposure to the progress of polymerization and gelation. The microrheological setup enabled a detailed study of three-dimensional gelation profiles; other experimental parameters that were initially varied include photoinitiator concentration, monomer composition, and light intensity. Significant changes in gelation time were observed with varying UV intensity and UV penetration depth into the sample. In addition, oxygen inhibition was found to significantly impact the cure speed of monomeric resins. The preliminary results were used to test the accuracy of the energy threshold model, which is often used to empirically predict the outcome of photopolymerization reactions. By using lithographic masks to generate well-defined UV illumination patterns with characteristic dimensions of tens of micrometers, it could be shown unambiguously that the diffusion of oxygen, an inhibitor, plays a critical role in the polymerization reaction. The experiments are in excellent agreement with a simple two-step model of oxygen consumption followed by polymerization. The use of high-speed electronic shutters in the UV light path enabled us to control the illumination time of the samples with high precision. Microrheological analysis could be used to reconstruct three-dimensional profiles of partially polymerized samples. Traditional photorheometry is not capable of resolving the evolution of sample rheology with such spatial resolution. In addition, experiments with pulsed illumination were used to quantify the role of dark reactions due to residual free radicals after termination of UV illumination.

In this thesis, I present a detailed analysis of the role cell morphology, solution rheology, and swimming mechanism has on the motility of Helicobacter Pylori. H. Pylori, the bacterium that causes gastric ulcers, has a helical cell shape that has long been believed to provide an advantage in penetrating the viscous mucus layer protecting the stomach lining, its niche environment. I present results obtained by performing optical microscopic live cell bacteria tracking of wild-type H. Pylori and cell shape and flagella mutants of H. Pylori. Bacteria tracking experiments show that helical shaped bacteria swim faster than straight rod-shaped bacteria, and bacteria with larger number of flagella swim faster. Altering cell shape is found to have a smaller effect on swimming speed than altering the number of flagella a bacterium has. These experimental observations are then compared to resistive force theory predictions. Resistive force theory shows qualitative agreement to our experimental observations, but overestimates the increase in swimming speed for a helical cell when compared to straight rod cell. In addition to effect of cell morphology on motility, I explore how motility is altered in different polymer environments by tracking bacteria in pig gastric mucin, methylcellulose, and gelatin solutions and gels. Bacteria are found to increase their swimming speed non-monotonically with increasing polymer concentration, while the number of mobile bacteria is found to decrease with increased polymer concentration. I also present an analysis of the swimming mechanism used by H. Pylori. H. Pylori is found to use a run-reverse swimming mechanism which I model as a random walk. This random walk model fits well to the experimental data and provides a theoretical tool for interpreting H. Pylori’s swimming mechanism. Taken together these results provide a detailed description of the motility of H. Pylori in different media and are applicable to the broad question of how H. pylori infects and colonizes the mucus layer of the stomach.

The goal of this thesis was to get familiar with the basics of mathematical description of the thermal motion of particles in a given media, and with other possibilities of the software package COMSOL Multiphysics. A model for viscous and viscoelastic environments was created, a uniform and user friendly system for simulation and calculation of MSD and system for data conversion from FCS to MSD. Furthermore, the possibilities of the model for use in microrheology were assessed and another procedure in the implementation of the COMSOL program in the characterization of gels was proposed.

A new method for determining the viscoelastic properties of materials was introduced and investigated. Results of three groups of samples obtained using one particle microrheology method, DLS microrheology method and classical rheology method were compared to be sure of correctness of measurements. As a model system were chosen mixtures of glycerol of different viscosities. In case of samples containing glycerol, results were also compared with tabulated values. Hyaluronan of various molecular weights was used as a biopolymer and polystyrene particles were used for microrheology. It was confirmed, that viscosity values of biopolymer samples obtaining by each method are comparable.

This thesis addresses development of a new force spectroscopy tool, correlation force spectroscopy (CFS), for the measurement of the mechanical properties of very small volumes of material (molecular to µm³) at kHz-MHz time-scales. CFS is based on atomic force microscopy (AFM) and the principles of CFS resemble those of dual-trap optical tweezers. CFS consists of two closely-spaced micro-cantilevers that undergo thermal fluctuations. Measurement of the correlation in thermal fluctuations of the two cantilevers can be used to determine the mechanical properties of the soft matter, e.g. a polymeric molecule, that connects the gap between the two cantilevers. Modeling of the correlations yields the effective stiffness and damping of the molecule. The resolution in stiffness is limited by the stiffness of the cantilever and the frequency by the natural frequency of the cantilevers, but, importantly, the damping resolution is not limited by the damping of the cantilever, which has enabled high-resolution measurements of the internal friction of a polymer. The concept of CFS was originally presented by Roukes' group in Caltech [Arlett et al., Lecture Notes in Physics, 2007]; I developed the first practical versions of CFS for experimentation, and have used it in two applications (1) microrheology of Newtonian fluids and (2) single molecule force spectroscopy. To understand the correlation in thermal fluctuations of two cantilevers I initially validated the theoretical approach for analyzing correlation in terms of deterministic model using the fluctuation-dissipation theorem [Paul and Cross, PRL, 2004]. I have shown that the main advantages of such correlation measurements are a large improvement in the ability to resolve stiffness and damping. Use of CFS as a rheometer was validated by comparison between experimental data and finite element modeling of the deterministic vibrations of the cantilevers using the known viscosity and density of fluids. Work in this thesis shows that the data can also be accurately fitted using a simple harmonic oscillator model, which can be used for rapid rheometric measurements, after calibration. The mechanical properties of biomolecules such as dextran and single stranded DNA (ssDNA) are also described. CFS measurements of single molecule properties of ssDNA reveal the internal friction of the molecule in solution. / Ph. D.