Aim: 

An optical tweezers setup consisting in two counter-propagating lasers (S. B. Smith et al., Science271, 795, 1996) is described. The setup, that allows to record forces up to 200 pN and length changes up to 100 mm, is aimed at studying the mechanics of single molecules, motor proteins and filaments. The application proposed here is for studying the interaction of the ensemble of myosin motors from a half-thick filament with the actin filament.

Methods and Results: 

Two microscope objectives face each other and focus two separate laser beams (l 830 nm, power 200 mW) to the same spot in a flow-through microchamber. The position of the microchamber is controlled by a low-profile closed-loop XYZ piezoelectric stage between the objectives. The beams partially fill the back aperture of the objectives and the photons leaving the trap are projected onto position-sensing photodiodes. The force on the bead is measured by the change in the light momentum. The design of the microchamber allows to have a laminar flow for stepwise change of the medium. The myosin filament is attached to the tip of a glass micropipette carried by the piezo manipulator that represents the length transducer. The bead-tailed actin, fluorescently labelled with phalloidin-rhodamine, is trapped in the focal point of the lasers and the actin filament is kept parallel to the myosin filament by laminar flow. The paths of the two lasers and of a blue light are aligned to simultaneously trap and observe the objects on a CCD camera. Temperature in the microchamber is controlled in the range 4.5 – 50 °C, by pumping fluid at the desired temperature through copper jackets placed on the objectives. The implementation in our system, that opens a new series of investigations in single molecule mechanics, is the possibility to operate either in length clamp mode (feedback from the piezo manipulator) or in force clamp mode (feedback from the position sensing photodiodes).

Supported by MiUR, Ente Cassa di Risparmio di Firenze (2005. 1245) and ITB-CNR (Milano).