Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.ip76

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High-Resolution Dual-Trap Optical Tweezers with Differential Detection: Alignment of Instrument Components

Carlos Bustamante, Yann R. Chemla, and Jeffrey R. Moffitt

Adapted from Single-Molecule Techniques (eds. Selvin and Ha). CSHL Press, Cold Spring Harbor, NY, USA, 2008.


INTRODUCTION

Optical traps or "optical tweezers" have become an indispensable tool in understanding fundamental biological processes. Using our design, a dual-trap optical tweezers with differential detection, we can detect length changes to a DNA molecule tethering the trapped beads of 1 bp. By forming two traps from the same laser and maximizing the common optical paths of the two trapping beams, we decouple the instrument from many sources of environmental and instrumental noise that typically limit spatial resolution. The performance of a high-resolution instrument--the formation of strong traps, the minimization of background signals from trap movements, or the mitigation of the axial coupling, for example--can be greatly improved through careful alignment. This procedure, which is described in this article, starts from the laser and advances through the instrument, component by component. Alignment is complicated by the fact that the trapping light is in the near infrared (NIR) spectrum. Standard infrared viewing cards are commonly used to locate the beam, but unfortunately, bleach quickly. As an alternative, we use an IR-viewing charge-coupled device (CCD) camera equipped with a C-mount telephoto lens and display its image on a monitor. By visualizing the scattered light on a pair of irises of identical height separated by >12 in., the beam direction can be set very accurately along a fixed axis.


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 Cold Spring Harb ProtocHome page
C. Bustamante, Y. R. Chemla, and J. R. Moffitt
High-Resolution Dual-Trap Optical Tweezers with Differential Detection: Managing Environmental Noise
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