The objective of this project is the development of a near-field optical microscope for the imaging and identification of single, fluorescently- tagged proteins in their native membranes. Because of the diffraction limit of light, the resolution of traditional optical microscopy is not sufficient to resolve single proteins with typical sizes on the order of 10 nm. 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, kinetic and dynamic properties of many membrane proteins are largely unknown. We propose to develop a novel scanning probe technique that uses a laser-irradiated, sharply pointed metal tip to create a concentrated and localized light source at the end of the tip by an effect known as field enhancement. The light source will be raster- scanned over the surface of a membrane with fluorescently labeled MEMBI 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 labeled proteins. The performance of the instrument will be tested by studying human AE1 transmembrane proteins in red blood cells. Besides playing an important role in blood CO2 transport, AE1 is a prototype for studying how ion exchange proteins work. At a later stage, the instrument will be applied to study distributions of adhesion proteins (E-selectin) in endothelial cell membranes. The applicant has established collaborations with biochemists that will help with sample preparation and provide input to biological questions. The instrument will be applicable to studies of other functionally important membrane properties such as rafts and other proteins, including other ion exchange proteins, active transport proteins, and cell adhesion proteins. ? ?
| Höppener, Christiane; Beams, Ryan; Novotny, Lukas (2009) Background suppression in near-field optical imaging. Nano Lett 9:903-8 |
| Hoppener, Christiane; Novotny, Lukas (2008) Antenna-based optical imaging of single Ca2+ transmembrane proteins in liquids. Nano Lett 8:642-6 |
