title: High-Resolution "Fleezers": Dual-Trap Optical Tweezers Combined with Single-Molecule Fluorescence Detection.
abstract: Recent advances in optical tweezers have greatly expanded their measurement capabilities. A new generation of hybrid instrument that combines nanomechanical manipulation with fluorescence detection-fluorescence optical tweezers, or "fleezers"-is providing a powerful approach to study complex macromolecular dynamics. Here, we describe a combined high-resolution optical trap/confocal fluorescence microscope that can simultaneously detect sub-nanometer displacements, sub-piconewton forces, and single-molecule fluorescence signals. The primary technical challenge to these hybrid instruments is how to combine both measurement modalities without sacrificing the sensitivity of either one. We present general design principles to overcome this challenge and provide detailed, step-by-step instructions to implement them in the construction and alignment of the instrument. Lastly, we present a set of protocols to perform a simple, proof-of-principle experiment that highlights the instrument capabilities.
authors.affiliation: Center for the Physics of Living Cells & Center for Biophysics and Quantitative Biology, University of Illinois, Urbana, IL, USA.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Whitley
authors.affiliation: Department of Physics and Astronomy, Michigan State University, East Lansing, MI, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Comstock
authors.affiliation: Center for the Physics of Living Cells & Center for Biophysics and Quantitative Biology, University of Illinois, Urbana, IL, USA. ychemla@illinois.edu.
authors.forename: Yann R
authors.initials: YR
authors.lastname: Chemla
mesh_terms: D002138:Calibration; D004247:DNA / Q000737:chemistry / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D017403:In Situ Hybridization / Q000379:methods / Q000592:standards; D018613:Microscopy, Confocal / Q000379:methods / Q000592:standards; D008856:Microscopy, Fluorescence / Q000379:methods / Q000592:standards; D015345:Oligonucleotide Probes; D052898:Optical Tweezers; D055095:Optics and Photonics / Q000295:instrumentation / Q000379:methods; D000072760:Single Molecule Imaging / Q000295:instrumentation / Q000379:methods* / Q000592:standards
chemical_list: D015345:Oligonucleotide Probes; D004247:DNA
keywords: Confocal microscopy; FRET; Fleezers; Förster resonance energy transfer; Optical trapping; Optical tweezers; Single-molecule fluorescence