Ultrahigh-resolution optical trap with single-fluorophore sensitivity

Nat Methods. 2011 Apr;8(4):335-40. doi: 10.1038/nmeth.1574. Epub 2011 Feb 20.

Abstract

We present a single-molecule instrument that combines a time-shared ultrahigh-resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, we observed individual single fluorophore-labeled DNA oligonucleotides to bind and unbind complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, we clearly observed coincident angstrom-scale changes in tether extension. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (for example, single-base-pair stepping of DNA translocases) along with the detection of properties of fluorescently labeled protein (for example, internal configuration).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA / chemistry*
  • DNA, Complementary / chemistry*
  • Fluorescent Dyes*
  • Micromanipulation
  • Microscopy, Confocal / methods
  • Microscopy, Fluorescence
  • Optical Devices*

Substances

  • DNA, Complementary
  • Fluorescent Dyes
  • DNA