Diffusion and Flow on Microscopic Length Scales Studied with Fluorescence Correlation Spectroscopy

Abstract

The physics of fluids on microscopic lengthscales is fundamentally different from the one governing macroscopic fluid phenomena. On lengthscales up to a few micrometer, inertial forces of small particles and fluid elements are much weaker than friction and thermal forces. Consequently, fluids on microscopic length scales are characterized by laminar flow and diffusive mixing. This is of particular importance for miniaturized technical applications, such as microfluidic devices, and many biological systems. However, the exact behavior of many of these systems is not yet fully understood and difficult to predict, especially for complicated geometries or fluids containing large amounts of solutes and macromolecules (crowded solutions). Experimental methods for measuring diffusion and flow on microscopic lengthscales can significantly increase our knowedge on these systems. Fluorescence Correlation Spectroscopy (FCS), for example, can be used for measuring translational diffusion, rotational diffusion, and flow of small fluorescent particles or molecules in solution. Two model systems were investigated here, to study the physics of crowded solutions, using recently developed FCS approaches for rotational and translational diffusion measurements. The translational and rotational diffusion of the large protein aldolase was studied in Polyethylene Glycol (PEG) solutions for different PEG sizes and concentrations. For comparison, the macroscopic vicosity and the diffusion of small dye molecules was investigated in the same system. While all measurements indicated a similar dependence of the diffusion coefficients on PEG concentration, the extent of the reduction in the diffusion coefficients varied depending on the size of the molecule under investigation. The translational diffusion of small dyes, as well as the rotational diffusion of aldolase was less affected by an increase in PEG concentration than the macroscopic viscosity and translational diffusion of aldolase. As a second model system, the translational and rotational diffusion of the protein αB crystallin was investigated at concentrations up to 300 mg/ml. Crystallin showed a decrease in diffusion coefficients with increasing concentrations, similar to the one observed in the systems containing PEG as a crowder. Finally, flow profiles of aligned liquid crystals in microfluidic devices were studied using FCS. Importantly, a local increase in flow velocity could be observed around a defect structure in the liquid crystal for the first time. These results confirm the suitability of FCS for studying complicated crowded enviroments and microfluidic geometries, thereby contributing to a better understanding of these systems.