Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.D etermining the molecular details of the time evolution of complex multicomponent biological systems requires analysis at the single-molecule level because of their stochastic and heterogeneous nature. Ideally, such experiments would track simultaneously the composition of a biological system (bound ligands, factors, and cofactors) and the conformation of the individual molecules in real time. Single-molecule fluorescence methods, such as total internal reflection fluorescence (TIRF) microscopy, allow the observations of the compositional dynamics (through arrival of fluorescently labeled ligands, factors, or cofactors) and conformational dynamics (through FRET) of single-molecular species. However, these traditional singlemolecule methods are hindered by limitations in maximal fluorescent component concentrations (up to 50 nM) (1), limited simultaneous detection (two to three colors) (2-6), and low throughput (a few hundred molecules at most per experiment) (7). As such, the full potential of single-molecule fluorescence to investigate a range of biological problems under physiologically relevant conditions has not yet been harnessed.Zero-mode waveguides (ZMWs) are small metallic apertures patterned on glass substrates that overcome the concentration restrictions by optically limiting background excitation (8). Each ZMW consists of an ∼150-nm-diameter metallic aperture that restricts the excitation light to a zeptoliter volume, making possible experiments with near-physiological concentrations (up to 20 μM) of fluorescently labeled ligands (1). Previous advances in nanofabrication (9), surface chemistry (10), and detection instrumentation (11) have led to ZMW-based instrumentation capable of the direct observation of DNA polymerization (12), reverse transcription (13), processive myosin motion (14), and translation by the ribosome (15, 16) with multicolor single-molecule detection. However, this sophisticated technology has not been broadly available to the scientific community. Despite multiple efforts to develop ZMW instrumentation, the combined difficulties in fabrica...

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