Fluorescence enhancement on silver nanostructures: studies of components of ribosomal translation in vitro

摘要

Metallic particles, silver in particular, can significantly enhance the fluorescence of dye molecules in the immediate vicinity (5-20 nm) of the particle. This magnifying effect can be theoretically explained/predicted by considering the change of photonic mode density near the fluorophore due to coupling to the conducting surface. We are using this method to observe fluorescence from a single ribosomal particle in a project aimed at acquiring sequence information from the translating ribosome (NIH's $1000 Genome Initiative). Several quartz slides with silver nanostructures were made using electron beam lithography techniques. The structures were approximately 50 nm high silver tiles measuring 400-700 nm on the side, and were spaced differently over a total area of 1 mm x 1 mm on any given quartz slide. In a preliminary experiment, we coated this surface with the Alexa 647-labeled antibodies and collected single molecule images using the MicroTime 200 (PicoQuant) confocal system. We showed that the fluorescence intensity measured over the silver islands film was more than 100-fold higher than fluorescence from a comparable site on uncoated section of the quartz slide. No noticeable photobleaching was seen. The fluorescence lifetime was very short, about 200 ps or less (this is the resolution limit of the system). The method has great promise for investigations of biologically relevant single molecules.