FIONA in the trap: the advantages of combining optical tweezers and fluorescence

Abstract

Technologies for the manipulation of single molecules have reached the resolution for the measurement of nanometre and sub-nanometre displacements and piconewton forces. In parallel with manipulation techniques, an array of single-molecule fluorescence detection methods have been developed to measure with great precision the position and/or the orientation of single biomolecules, as well as their conformational fluctuations. A new generation of instruments devoted to single-molecule biophysics is now emerging from the combination of two or more single-molecule techniques into one set-up. Particularly fruitful is the combination of manipulation techniques with single-molecule fluorescence techniques, allowing the detection of biomolecule position, conformation or biochemical state simultaneously with the measurement (or the external control) of mechanical output. Here we present the combination of optical tweezers and fluorescence imaging with nanometre accuracy (FIONA). The apparatus was tested on an actin filament labelled with a quantum dot and suspended in solution in a dumbbell configuration using the laser tweezers. This apparatus allows control of the mechanical conditions of a track (actin, microtubules, nucleic acids) while monitoring, by fluorescence, locomotion (and, possibly, biochemical state) of a motor on the track, thus being applicable to a large variety of biological systems