CAREER: Tip-Enhanced Fluorescence Microscope for Resolving Single Molecules Within Dense Biomolecular Networks

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This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).The main objective of this project is to develop an optical microscope that is capable of deciphering the organization of intact biomolecular networks at the molecular length scale, with a particular emphasis on membrane-associated networks. The function of such a network is closely related to its structure, which can be disrupted by genetically or environmentally triggered conformational changes to the molecular components, sometimes resulting in disease or other malfunction. A key weakness of most high-resolution structural techniques for deciphering the network architecture is their inability to determine molecular composition, since they generally lack the sensitivity to identify individual molecules. Only optical spectroscopy can routinely achieve single-molecule detection, but traditional optical techniques are not sensitive enough to resolve the molecular features of a sample by almost two orders of magnitude. The technical component of this award involves a number of innovative improvements to an emerging high-resolution structural analysis technique called tip-enhanced fluorescence microscopy (TEFM). TEFM is a near-field optical method whereby the optical intensity of a laser beam is concentrated at the apex (tip) of a sharp needle analogous to a lightning rod. The enhanced-intensity region increases the fluorescence rate in the vicinity of the tip, which enables spatial resolution limited only by its sharpness. TEFM has been used to image individual molecules with ~10 nanometer spatial resolution, and is particularly promising for studying planar samples such as biological membranes. Several innovations in this project, including a polarization modulation scheme and customized single-photon analysis algorithms, increase TEFM contrast and improve compatibility with soft biological samples. To increase its versatility, TEFM combines on a single integrated platform with other imaging modalities such as wide-field fluorescence microscopy, confocal microscopy, phase contrast microscopy, total internal fluorescence microscopy, fluorescence correlation microscopy, and fluorescence lifetime microscopy. Several independent spectral channels are also implemented to enable simultaneous multi-color imaging. Finally, temperature regulation and closed-loop focus control will be developed for imaging live cell membranes. The instrumentation developed under this award is expected to have broad implications for biological imaging research. Results will be published in the scientific literature and also on a publicly accessible webpage. Detailed information will be shared upon request. The education and outreach component of this award involves the development of an undergraduate integrated science major; mixed-media modules for high-school students and college freshmen on the Physics of various outdoor activities (e.g. skiing, sailing, SCUBA, surfing); and science modules for elementary school students. These projects are ongoing and the results will be described on a publicly accessible webpage. Education materials developed under this award will be provided upon request.