High-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy

K. D. Whitley, M. J. Comstock, Y. R. Chemla

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages137-169
Number of pages33
DOIs
StatePublished - 2017

Publication series

NameMethods in Enzymology
Volume582
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Confocal microscopy
  • FRET
  • Förster Resonance Energy Transfer
  • Helicase
  • Optical trapping
  • Optical tweezers
  • Single-molecule fluorescence
  • UvrD

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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  • Cite this

    Whitley, K. D., Comstock, M. J., & Chemla, Y. R. (2017). High-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy. In Methods in Enzymology (pp. 137-169). (Methods in Enzymology; Vol. 582). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2016.10.036