Single molecule fluorescence studies of ribosome dynamics: An application of metal enhanced fluorescence

Metal enhanced fluorescence (MEF), in which a surface plasmon near a noble metal alters the spectral properties of an organic fluorophore, has been reported to increase fluorescence intensity without a concomitant increase in photobleaching rate. The fluorescence intensities of Cy3- and Cy5-labeled ribosomal initiation complexes (ICs) near 50 nm silver particles were increased 4 - 7-fold compared to ICs in the absence of silver colloids. Photobleaching lifetime was not significantly decreased, resulting in 4 - 5.5-fold enhancement in total photon emission prior to photobleaching. Fluorophores showing enhanced fluorescence were located within ∼280 nm of the colloidal particles, as detected by light scattering and scanning probe microscopy. Aggregates of silver particles or larger colloids themselves produced wavelength-shifted luminescence similar to fluorescence, presumably due to resonant extinction between nearby metal particles. Intensity fluctuations above shot noise, at 0.1 - 5 Hz, were greater from slides containing colloidal particles than from plain glass. Overall signal to noise ratio was similar or slightly better near the silver particles. Proximity to silver particles did not compromise ribosome function, as measured by codon-dependent binding of fluorescent tRNA to the A site of fluorescent labeled ribosomes, dynamics of fluorescence resonance energy transfer between adjacent tRNAs in the ribosomal A and P sites, and elongation factor G catalyzed translocation.