High-resolution “fleezers”: Dual-trap optical tweezers combined with single-molecule fluorescence detection

Kevin D. Whitley, Matthew J. Comstock, Yann R. Chemla

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages183-256
Number of pages74
DOIs
StatePublished - Jan 1 2017

Publication series

NameMethods in Molecular Biology
Volume1486
ISSN (Print)1064-3745

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Whitley, K. D., Comstock, M. J., & Chemla, Y. R. (2017). High-resolution “fleezers”: Dual-trap optical tweezers combined with single-molecule fluorescence detection. In Methods in Molecular Biology (pp. 183-256). (Methods in Molecular Biology; Vol. 1486). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6421-5_8