title: Dark-field illumination on zero-mode waveguide/microfluidic hybrid chip reveals T4 replisomal protein interactions.
abstract: The ability of zero-mode waveguides (ZMWs) to guide light energy into subwavelength-diameter cylindrical nanoapertures has been exploited for single-molecule fluorescence studies of biomolecules at micromolar concentrations, the typical dissociation constants for biomolecular interactions. Although epi-fluorescence microscopy is now adopted for ZMW-based imaging as an alternative to the commercialized ZMW imaging platform, its suitability and performance awaits rigorous examination. Here, we present conical lens-based dark-field fluorescence microscopy in combination with a ZMW/microfluidic chip for single-molecule fluorescence imaging. We demonstrate that compared to epi-illumination, the dark-field configuration displayed diminished background and noise and enhanced signal-to-noise ratios. This signal-to-noise ratio for imaging using the dark-field setup remains essentially unperturbed by the presence of background fluorescent molecules at micromolar concentration. Our design allowed single-molecule FRET studies that revealed weak DNA-protein and protein-protein interactions found with T4 replisomal proteins.
authors.affiliation: Department of Engineering Science and Mechanics and ‡Department of Chemistry, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Zhao
authors.forename: Danqi
authors.initials: D
authors.lastname: Chen
authors.forename: Hongjun
authors.initials: H
authors.lastname: Yue
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Spiering
authors.forename: Chenglong
authors.initials: C
authors.lastname: Zhao
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
authors.forename: Tony Jun
authors.initials: TJ
authors.lastname: Huang
mesh_terms: D017122:Bacteriophage T4 / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004867:Equipment Design; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation; D008027:Light; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D038761:Photobleaching; D025941:Protein Interaction Mapping / Q000295:instrumentation*; D059629:Signal-To-Noise Ratio; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins