The dynamic behavior of biomolecules at surfaces and interfaces is of fundamental importance to many applications, including biosensors, pharmaceutical processing and delivery, biomedical devices and consumer products. We have developed high-throughput single molecule tracking methods based on single molecule total internal reflectance fluorescence (smTIRF) microscopy to characterize molecular dynamics at interfaces. Unlike ensemble averaged techniques, single molecule tracking can characterize the full ensemble of dynamic behavior. We have discovered a rich diversity of dynamic behavior, including multiple modes of diffusion associated with molecular conformation, distinct molecular populations with characteristic surface residence times and diffusion, interfacial protein aggregation and many more. In this talk, I will describe two systems we have investigated using smTIRF microscopy: proteins at the oil-water interface and fatty acids at solid-liquid interface. The dynamics of isolated individual protein objects at the oil-water interface revealed multiple protein populations associated with aggregate states (monomer, dimer, trimer). Adsorption of isolated proteins was found to be reversible, with desorption rates correlated to aggregate state of the protein object. Single molecule tracking was also used to characterize the initial stages of interfacial protein aggregation and subsequent protein film formation. Using our high-throughput single molecule tracking, we have developed a new super-resolution chemical imaging method, MAPT (Mapping using Accumulated Probe Trajectories). MAPT analysis generates spatial maps of absolute physical quantities (adsorption rate, desorption probability, local surface diffusion coefficient, surface coverage/occupancy) that are directly associated with the molecular interactions between a probe molecule and the surface. Unlike other chemical imaging methods, MAPT excels at characterizing “soft” molecular materials, for which “wet” conditions and non-covalent interactions are important. MAPT images of fatty acid interactions with patchy hydrophobic surfaces have revealed a new “flying” (desorption-mediated) mode of surface diffusion that is the primary transport mode associated with the formation of self-assembled monolayers. ____________________________________________________________________________ Robert Walder is a post-doctoral associate in the Department of Chemical and Biological Engineering at the University of Colorado at Boulder. He received his Ph.D. degree in 2008 from the Department of Physics at the University of California, Irvine, under the supervision of Prof. Michael Dennin.