The drive toward more sensitive single-molecule manipulation techniques has led to the recent development of optical tweezers capable of resolving the motions of biological systems at the subnanometer level, approaching the fundamental limit set by Brownian fluctuations. One successful approach has been the dual-trap optical tweezers, in which the system of study is held at both ends by microspheres in two separate optical traps. We present here a theoretical description of the Brownian limit on the spatial resolution of such systems and verify these predictions by direct measurement in a Brownian noise-limited dual-trap optical tweezers. We find that by detecting the positions of both trapped microspheres, correlations in their motions can be exploited to maximize the resolving power of the instrument. Remarkably, we show that the spatial resolution of dual optical traps with dual-trap detection is always superior to that of more traditional, single-trap designs, despite the added Brownian noise of the second trapped microsphere.single molecule ͉ subnanometer resolution ͉ signal-to-noise ratio S ince the discovery that optical gradients could stably trap micrometer-sized dielectric particles (1), gradient optical traps, or optical tweezers, have been used in a large variety of applications ranging from microfabrication (2, 3) to the study of colloidal hydrodynamics (4-6) and nonequilibrium thermodynamics (7-10). In particular, the use of calibrated optical springs has provided unprecedented new insight on the mechanical properties of single biological molecules and the molecular motors that manipulate them in the cell (11)(12)(13)(14). In a typical single-molecule experiment, a biopolymer is linked to a single, optically trapped dielectric microsphere at one end and to a fixed surface at the other. The length of this molecule is inferred from the force applied by the optical trap and the position of the microsphere relative to the fixed end of the polymer. In an ideal system with no measurement error, the ability to resolve changes in the length of the polymer, the spatial resolution of the optical tweezers, is limited only by the stochastic Brownian force induced on the microsphere by the surrounding solvent. This limit has been estimated previously (15,16) and implies that it should be possible to resolve length changes on the angstrom scale with reasonable values of the experimental parameters, i.e., microsphere size, polymer stiffness, and averaging bandwidth. However, it has proven difficult to sufficiently isolate single-trap optical tweezers from environmental and instrumental sources of noise to reach this Brownian noise limit. Drift and low frequency fluctuations of the fixed surface relative to the optical trap typically obscure these small movements.One method to increase the stability of optical tweezers is to introduce a second optical trap to hold the other end of the system, thereby decoupling it from movements of the fixed surface. Although such dual-trap optical tweezers were first envisioned m...