title: High-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy.
abstract: We describe the design, construction, and application of an instrument combining dual-trap, high-resolution optical tweezers and a confocal microscope. This hybrid instrument allows nanomechanical manipulation and measurement simultaneously with single-molecule fluorescence detection. We present the general design principles that overcome the challenges of maximizing optical trap resolution while maintaining single-molecule fluorescence sensitivity, and provide details on the construction and alignment of the instrument. This powerful new tool is just beginning to be applied to biological problems. We present step-by-step instructions on an application of this technique that highlights the instrument's capabilities, detecting conformational dynamics in a nucleic acid-processing enzyme.
authors.affiliation: University of Illinois at Urbana-Champaign, Urbana, IL, United States.
authors.forename: K D
authors.initials: KD
authors.lastname: Whitley
authors.affiliation: Michigan State University, East Lansing, MI, United States.
authors.forename: M J
authors.initials: MJ
authors.lastname: Comstock
authors.affiliation: University of Illinois at Urbana-Champaign, Urbana, IL, United States; Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL, United States. Electronic address: ychemla@illinois.edu.
authors.forename: Y R
authors.initials: YR
authors.lastname: Chemla
mesh_terms: D004265:DNA Helicases / Q000737:chemistry / Q000302:isolation & purification*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D036103:Nanotechnology / Q000379:methods; D052898:Optical Tweezers; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D004265:DNA Helicases
keywords: Confocal microscopy; FRET; Förster Resonance Energy Transfer; Helicase; Optical trapping; Optical tweezers; Single-molecule fluorescence; UvrD