Figure 4.

Enhanced control using two force-measuring optical traps. (A) Schematic of the dual-trap assay. Two traps can be generated from a single laser source by splitting into two orthogonal polarizations, which may be independently steered in the sample. After suspending a single DNA molecule in between two trapped beads, the DNA can be manipulated without the application of force. In addition, optical tweezers can be employed to quantitatively detect the forces exerted on the DNA. The fluorescence from DNA-staining dyes or fluorescently labeled DNA-binding proteins can be detected using a CCD camera. (B) The assay from panel a employed in our study of the elasticity of (fluorescently labeled) RAD51 nucleoprotein filaments formed on double-stranded DNA (50). One DNA molecule is suspended between two optically trapped beads (dark circles); a second molecule is tethered from the lower bead and freely diffusing once buffer flow is switched off (between 2nd and 4th frame). By increasing the distance between the traps, tension can be applied to the suspended DNA in a controlled manner. The differentiated extension of the fluorescent, RAD51-coated segments and the dark, uncoated segments can be directly seen. The increasing suppression of thermally excited diffusion of the DNA is readily observed.