Single molecule and nanoparticle imaging has become a very important tool to investigate many biological and chemical processes. This dissertation presents the applications of single molecule and nanoparticle imaging in biophysical, surface, and photocatalysis studies using far-field optical microscopy. The work was mainly carried out under a differential interference contrast (DIC) microscope and a total internal reflection (TIR) microscope.

First, a DIC microscopy-based single particle orientation and rotational tracking technique that allows for resolving the full three-dimensional (3D) orientation of single gold nanorod (AuNR) probes has been developed. The angular degeneracy was overcome by combining DIC polarization anisotropy with the image pattern recognition technique. The usefulness of this technique in biophysical studies was further verified by real time tracking of rotational motions of single AuNRs rotating on live cell membranes. Therefore, it is expected that this method will enable us to elucidate the comprehensive interaction mechanisms between the functionalized nanocargoes and the membrane receptors in live cells. Detailed in situ conformational information on how they bind on the cell membrane and how they move and rotate in live cells at single particle level would provide new avenues for the development of new generation of high efficient drug and gene delivery carriers.

Second, a high-throughput focused orientation and position imaging (FOPI) technique with 3D orientation resolvability for single AuNRs deposited on a gold film has been developed for surface studies. The FOPI method presented in this dissertation provides a new approach using the interaction of AuNRs with their surrounding environment for resolving the 3D orientation of single AuNRs. Therefore, it is expectedthat this method can be used as a tool to study interactions of functionalized nanoparticles with functional gold surfaces.

Last, single molecule TIRF imaging was successfully employed in photocatalysis study to reveal the nature and photocatalytic properties of the surface active sites on single Au-CdS hybrid nanocatalysts. Single-molecule photocatalysis with high-resolution super-localization imaging allowed us to reveal two distinct, incident energy-dependent charge separation mechanisms in single Au-CdS heterostructures. This finding will help us design and develop better metal-semiconductor heterostructures that are highly active for photocatalytic reactions under visible light. Furthermore, the finding will enable us to potentially engineer the direction of energy flows on the heterostructured nanomaterials at the nanoscale. Therefore, it is expected that the results presented in this dissertation have a potential impact on the development of better photocatalyst structures.