Lilliputian optics: Theory, experiment, and applications of nanoscale materials for biological microscopy

Optical microscopy is a powerful tool for biology, characterized by the attainable spatial and temporal imaging resolution. This thesis explores the theoretical understanding and experimental application of nanoscale materials to the improvement of both spatial and temporal resolution for biological application.Chapter I provides the background, describing the limitations on far-field spatial resolution, the means for breaking the diffraction limit, and requirements for high time resolution imaging.Chapter II describes finite difference time domain (FDTD) based design of tip enhanced nonlinear optical microscopy (TENOM) probes for optimal field enhancement, facilitating high spatial resolution optical imaging. Accurate calculation of the electric field enhancement by nanoscale gold structures is achieved using the modified Debye model to fit the measured optical constants of gold. Analytical and FDTD calculations demonstrate that probes on the scale of the excitation wavelength outperform quasi-infinite probes, and that trigonal pyramidal probes yield particularly large enhancements.Chapter III describes focused ion beam (FIB) based fabrication of TENOM probes ranging from sharpened cones to finite trigonal pyramids and probes for the reduction of fluorescence quenching. The combination of sub-10 nm spatial resolution milling, nanometric deposition of dielectric materials, and micromanipulation capabilities makes the dual-beam FIB a uniquely powerful tool for probe fabrication. These probes are used for high resolution imaging experiments.Chapter IV addresses the use of FDTD calculations for the characterization of fluorescence quenching by TENOM probes. Quenching by TENOM probes is vastly different than quenching by mirrors. Signal enhancement is calculated by combination of field enhancement and quenching simulations. These calculations show that the halo-type images seen in TENOM are likely caused by quenching, and that high field enhancement or separation of the probe from the fluorophore by &sim10 nm can alleviate the problem.Finally, Chapter V examines the use of quantum dots for high temporal resolution imaging in vivo. Their brightness and photostability make it possible to record images on the 7 ms/frame scale, though blinking is a significant complication. Blinking is found to be strongly suppressed in live cells. The cause of in vivo blinking suppression is explained through a ligand binding molecular orbital model.