Protocol

Optical Tweezers

Volume 1486 of the series Methods in Molecular Biology pp 183-256

Date:

High-Resolution “Fleezers”: Dual-Trap Optical Tweezers Combined with Single-Molecule Fluorescence Detection

  • Kevin D. WhitleyAffiliated withCenter for the Physics of Living Cells & Center for Biophysics and Quantitative Biology, University of Illinois
  • , Matthew J. ComstockAffiliated withDepartment of Physics and Astronomy, Michigan State University
  • , Yann R. ChemlaAffiliated withCenter for the Physics of Living Cells & Center for Biophysics and Quantitative Biology, University of Illinois Email author 

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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.

Key words

Optical tweezers Optical trapping Single-molecule fluorescence Förster resonance energy transfer FRET Confocal microscopy Fleezers