This proposal seeks to fundamentally improve the resolution of optical imaging by exploiting the properties of optical antennas, a new concept in the field of optical physics. The objective is to develop an instrument for the imaging, identification and characterization of single proteins in native cell membranes. An instrument with such capability will have a high impact on various fields of biological research because membrane proteins undergo a multitude of specific tasks ranging from recognition and signaling to molecular transport across the cell membrane. Although current methods, such as X-ray crystallography or NMR, provide high-resolution structural details, they are limited by their static observation and ensemble averaging. As a consequence, the spatial distribution and co-localization of membrane proteins as well as the correlations with membrane topology are largely unknown. The instrument to be developed in this project uses an optical antenna to localize and enhance incident laser radiation thereby creating a nanoscale light source. This light source will be rasterscanned over the surface of a biological membrane with fluorescently labeled proteins. Each point on the sample surface will be characterized by its unique fingerprint in form of a fluorescence spectrum allowing us to localize, identify and study single proteins. Preliminary results support the feasibility of this technique but various issues need yet to be addressed. We will use fused gold nanoparticles and gold nanorods as optical half-wave antennas and attach them to pointed dielectric capillaries. Different approaches for suppression of background fluorescence originating from the cell interior will be investigated. The instrument will be used to study distributions of adhesion proteins in endothelial cell membranes which play a key role in leukocyte-endothelial cell interactions and thus in the inflammatory response. Single layers of endothelial cells will be grown directly on functionalized glass substrates and P-selectin proteins will labeled with fluorescent antibodies. The goal is to spatially resolve individual proteins under physiological conditions and to study the correlation with membrane topology.
