Investigation of Small Molecule Diffusion in Complex Fluids Using Fluorescence Correlation Spectroscopy

Complex fluids are a class of liquids whose microscopic heterogeneity has proven advantageous in several biological and industrial applications. While rheological and structural characterization studies have been the primary forms of examining complex fluids, the translational diffusion behavior of small molecules within these systems has been less explored. Fluorescence correlation spectroscopy (FCS) is a useful technique for measuring translational diffusion rates of small molecules based on fluorescence intensity fluctuations arising from fluorophores moving through a small volume of focused laser light. Here, an FCS instrument utilizing two-photon excitation has been fully constructed and calibrated, and the examination of small molecule dynamics within two classes of complex fluids, bicontinuous microemulsions and giant unilamellar vesicles, using FCS will be presented. In the bicontinuous microemulsion study, specific focus was given to the impact of surfactant structure on small molecule diffusion. It was concluded that the length of the head group and overall size and bulkiness of the surfactant in the interfacial layer provided the largest impact on probe diffusion. For the GUV studies, branched chain fatty acids present in bacterial cellular membranes are suspected to increase the fluidity of the lipid bilayer much like unsaturated lipids do in mammalian cells. FCS results have revealed that GUVs containing a branched chain fatty acid display higher fluidity than membranes with unsaturated lipids. While preliminary results have been obtained for the GUVs, further studies minimizing artifacts and incorporating additional bacterial membrane specific components are still needed to obtain a more cohesive picture of bacterial membrane dynamics.