The primary objectives of the proposed work are to develop improved methods of single-biomolecule optical detection and spectroscopy, and to apply these methods to several key biological problems of importance. The important advantage of the single-molecule regime is that signatures of conformational state and dynamical changes which are normally buried in ensemble experiments will become uniquely accessible to study. The development of techniques for single-biomolecule optical imaging and spectroscopy rests upon a balanced combination of (i) enhancement and extension of established techniques driven by specific biological applications, and (ii) exploratory investigation of new detection modalities. A new type of microscopy based on scanning of electromagnetically enhanced metal structures will be developed, which should lead to higher sensitivities as well as higher resolution. Specific biological problems of importance will be used to challenge the techniques, in order to stimulate the development of new capabilities. The principal biological problems of interest are: (a) detection and imaging of the diffusion of single copies of major histocompatibility complexes of type II in living cells in the presence of varying membrane cholesterol concentrations, binding proteins, and during T-cell receptor interactions, (b) measurements of the single-molecule enzymatic behavior of beta-galactosidase with an aim to explore the underlying stochastic dynamics in the absence or presence of various inhibitors, and (c) the observation and understanding of assisted protein folding in single bacterial and eukaryotic chaperones using a variety of reporter fluorophore technologies. To accomplish these ends, the Pl's physical, chemical, and optical expertise will be combined with the molecular biological, biochemical, and synthetic expertise of several collaborators. In the final analysis, owing to its multi-disciplinary organization, the fundamental research in this program will not only generate new knowledge about the biophysical properties of several important protein systems at the single-molecule level, but, in addition the advances in instrumentation will provide novel groundwork for technology transfer to other relevant disciplines.