Characterizing Electrostatic Interactions at Interfaces using Quantitative Single Molecule Fluorescence Microscopy

Rather than using single-molecule fluorescence microscopy to make simple structural maps, this research is focused on developing advanced imaging methods to make quantitative measurements of chemical behavior at interfaces. We have adapted single-molecule techniques to make quantitative measurements of the influence of electrostatics on concentrations, equilibria, and kinetics based on the stochastic behavior of molecules at solid-liquid interfaces. To enable these efforts, we developed algorithms to quantitatively measure single molecule populations in noisy images, enabling us to count fluorescent molecules using minimal fluorescence excitation intensity to minimize photobleaching and photoblinking that complicate tracking molecule trajectories. This methodology was used to study fluorescently labeled DNA plasmids interacting with an electrically polarized interface. By tracking diffusing DNA in the interface, we quantitatively measured the population and diffusion coefficient of solution-phase DNA in the diffuse double-layer near a transparent semiconductor-solution interface, and used a model based on Gouy-Chapman theory to estimate the net electrical charge of DNA screened by counterions. This quantitative single-molecule methodology also allowed us to measure the kinetics and equilibria of reversible hybridization between short solution-phase DNA fragments and surface-bound complementary oligonucleotides. Persistence lifetimes of probe DNA in the hybridized state give a direct quantitative measurement of the double-helix dissociation rate constant. The surface density of target DNA and the association constant of the bound complex were quantified by counting molecules in fluorescence images and using this absolute surface density to calibrate a Langmuir isotherm based on fluorescence intensity. We measured the impact of increasing buffer ionic strength on hybridization kinetics and equilibria, and used these data to inform a structure-based electrostatic model for double-helix formation. This model was able to estimate the energetic contribution of electrostatic repulsion and the number of interacting electrical charges in the double-helix transition state and fully hybridized state.