No More “Wiggles”: Decoherent Acousto Optic-Based High-Resolution Tweezers Combined with Multi-Color Fluorescence

Abstract

Recent advances in hybrid single molecule methods that couple angstrom-resolution optical trapping with single molecule fluorescence (high-resolution fleezers) have demonstrated the utility of measuring protein activity and conformation simultaneously e.g., revealing detailed helicase conformation coupled to DNA unwinding. Here we present advancements in multi-color high-resolution fleezers methods. First, we have developed a high-resolution fleezers instrument with three-color confocal fluorescence microscopy. To reduce photobleaching caused by the optical traps, timeshared dual optical traps were interlaced and synchronized with three fiber coupled fluorescence excitation lasers (473 nm, 532 nm, and 633 nm) and three single-photon counting fluorescence detectors. We report performance with a set of fluorophores varying both optical trap and fluorescence excitation intensity. In the fleezers, the Cy5 fluorophore dramatically outperforms other commonly used fluorophores. Second, we have devised a method that completely removes acousto-optic device trap positioning errors. Acousto-optic devices have been applied extensively in optical tweezers because of their flexibility and speed, however these devices have well-known trap positioning inaccuracies (‘wiggles’) that limit their usefulness, particularly for high-resolution applications. We show that these inaccuracies are due to interference patterns within the device sound fields. We have devised a method that removes these inaccuracies by reducing the coherence of the sound fields by directly controlling and randomizing the phase of the radio frequency control. We demonstrate that the trapping inaccuracies are completely eliminated, and that no additional measurement noise is added, for both constant trap position and force-ramp measurements. We show that this random phase method is applicable both to acousto-optic modulator and deflector type devices and can be easily integrated via a software upgrade into existing instruments.