title: High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence.
abstract: 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.
authors.affiliation: Department of Applied Physics, Stanford University, Stanford, CA 94305.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.forename: Ravindra V
authors.initials: RV
authors.lastname: Dalal
authors.forename: Alexey N
authors.initials: AN
authors.lastname: Petrov
authors.forename: Albert
authors.initials: A
authors.lastname: Tsai
authors.forename: Seán E
authors.initials: SE
authors.lastname: O'Leary
authors.forename: Karen
authors.initials: K
authors.lastname: Chapin
authors.forename: Janice
authors.initials: J
authors.lastname: Cheng
authors.forename: Mark
authors.initials: M
authors.lastname: Ewan
authors.forename: Pei-Lin
authors.initials: PL
authors.lastname: Hsiung
authors.forename: Paul
authors.initials: P
authors.lastname: Lundquist
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Turner
authors.forename: David R
authors.initials: DR
authors.lastname: Hsu
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Puglisi
mesh_terms: D000465:Algorithms; D001703:Biophysics / Q000295:instrumentation / Q000379:methods*; D003199:Computer Systems; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D008991:Monitoring, Physiologic / Q000295:instrumentation / Q000379:methods*; D012984:Software