Characterization of Industrial and Biological Complex Fluids Using Confocal Microscopy.

Complex fluids are materials that respond to applied stresses in a way that is intermediate between that of a purely viscous fluid or a fully elastic solid. The characterization of material properties depends upon the ability to both apply variable stresses or strains to the complex fluid and to measure the resulting response. Often the macroscopic response of these materials depends upon the microscale structure. In this dissertation, confocal laser scanning microscopy is used to analyze microscopic changes in complex fluids. We developed methods to apply the stress or strain that matches the application or environment of the material to be characterized. For instance, confocal images of the emulsion structure of fountain solution in ink exposed to oscillatory shear flow on model substrates were acquired. We found that surfactants inhibit aqueous droplet wetting on hydrophobic substrates. Without surfactants, surface coverage of the aqueous fountain solution on the hydrophobic substrate became quite high. This result is relevant to defects in lithographic printing. To characterize the material properties of biofilms a tunable small-scale device was needed. We developed a flexible microfluidic rheometer to apply a compressive force of ~200 pL volume. We used confocal microscopy to detect the deformation of a membrane in contact with a test material when compressive stresses were applied. This measurement allowed us to characterize material properties including elastic modulus and relaxation time of soft viscoelastic solids, biofilms in particular. We report evidence of strain hardening in biofilms; a result that could have implications for the understanding of clearance of biofilms in industrial and physiological environments. To understand the source of this phenomenon we applied confocal microscopy to characterize the structure of bacteria aggregated in a polysaccharide matrix. Interestingly, while aggregated bacteria and bacteria in biofilms are held together by ostensibly the same material we find their inter-bacterial distance to be quite different. Once this tool was developed the origin of strain-hardening in biofilms could be addressed. The strain-induced trajectories of individual bacteria found using image processing of confocal micrographs were analyzed to show that strain hardening occurs without an increase in volume fraction.Ph.D.Chemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/61778/1/dnhohne_1.pd

Contribution of the Klebsiella pneumoniae Capsule to Bacterial Aggregate and Biofilm Microstructures▿ †

We studied the interaction between capsule production and hydrodynamic growth conditions on the internal and macroscopic structure of biofilms and spontaneously formed aggregates of Klebsiella pneumoniae. Wild-type and capsule-deficient strains were studied as biofilms and under strong and mild hydrodynamic conditions. Internal organization of multicellular structures was determined with a novel image-processing algorithm for feature extraction from high-resolution confocal microscopy. Measures included interbacterial spacing and local angular alignment of individual bacteria. Macroscopic organization was measured via the size distribution of aggregate populations forming under various conditions. Compared with wild-type organisms, unencapsulated mutant organisms formed more organized aggregates with less variability in interbacterial spacing and greater interbacterial angular alignment. Internal aggregate structure was not detectably affected by the severity of hydrodynamic growth conditions. However, hydrodynamic conditions affected both wild-type and mutant aggregate size distributions. Bacteria grown under high-speed shaking conditions (i.e., at Reynolds' numbers beyond the laminar-turbulent transition) formed few multicellular aggregates while clumpy growth was common in bacteria grown under milder conditions. Our results indicate that both capsule and environment contribute to the structure of communities of K. pneumoniae, with capsule exerting influence at an interbacterial length scale and fluid dynamic forces affecting overall particle size