Project Summary/Abstract Our research program aims to directly probe how the complexity of living bacterial membranes impacts the adsorption, transport, and domain association of small molecules, including antibiotics. To address these points, we will leverage nonlinear spectroscopy and microscopy techniques, specifically second harmonic generation, to map the dynamic behavior. A key to our methodology is the ability to conduct the proposed experiments on living cells instead of model systems. For the next 5 years, our program goals are to (1) extract the key factors that influence the adsorption and membrane organization of small molecule membrane probes, (2) quantitatively assess the adsorption of tetracycline antibiotics and manipulate their movement within and through the membranes of different species of bacteria, and (3) examine the spatial dependence of small molecule-membrane interactions on individual bacteria as well as within biofilms. Together these studies will elucidate the role of how parameters including curvature, membrane domains, and the cell wall mediate small molecule uptake. We envision that this insight will provide new directions in the continued pursuit of improved antibiotics.