Development of near-field studies of single biomolecules.

Abstract

Many problems in contemporary biological chemistry require a detailed description of the underlying molecular mechanics of biological processes. Unfortunately, traditional techniques which employ large populations of molecules only obtain information on the average behavior of the molecular ensemble. Averaged information often obscures important details of molecular structure and conformational changes. Recently developed methodologies to study single biomolecules based on single fluorophore detection avoid ensemble averaging problems and provide greater detail on molecular structure and mechanics in vitro. Diffraction limits the resolution of fluorescence based methodologies to $>$200 nm for visible light which prevents direct structural investigations of single biomolecules as well as studies of the behavior of single biomolecules in high density in situ or in vivo settings. We have developed a new spectroscopic and imaging approach to address this problem based on the technique of near-field scanning optical microscopy (NSOM), a methodology for obtaining sub-diffraction limited resolution (20-200 nm) with the spectroscopic information afforded by conventional optical techniques. This thesis presents an experimental demonstration of the development of a NSOM system capable of studying single biomolecules using single fluorophore detection. Near-field probe manufacturing procedures and NSOM system design are discussed. We have detected and imaged the fluorescence from single fluorophores with a resolution below the diffraction limit. We have also investigated the application of two-photon induced fluorescence to the imaging of single fluorophores. Compared to linear excitation, two photon excitation resulted in an improved spatial resolution for uncoated near-field probes but also in a reduced photostability of the fluorophores. Additionally, we have developed a special liquid cell sample holder to overcome the technical difficulties associated with NSOM imaging of soft biological samples under water. With this development and the demonstrated single fluorophore detection sensitivity, the tools exist to extend near-field microscopy to studies of single biomolecules in an aqueous environment. Methodologies for using NSOM to study single biomolecules are discussed and potential experiments are identified.