  Dual trap optical tweezers are often used in high-resolution measurements in
single-molecule biophysics. Such measurements can be hindered by the presence
of extraneous noise sources, the most prominent of which is the coupling of
fluctuations along different spatial directions, which may affect any optical
tweezers setup. In this paper we analyze, both from the theoretical and the
experimental points of view, the most common source for these couplings in Dual
Trap Optical Tweezers setups: the misalignment of traps and tether. We give
criteria to distinguish different kinds of misalignment, to estimate their
quantitative relevance and to include them in the data analysis. The
experimental data is obtained in a novel dual trap optical tweezers setup which
directly measures forces. In the case in which misalignment is negligible we
provide a method to measure the stiffness of traps and tether based on variance
analysis. This method can be seen as a calibration technique valid beyond the
linear trap region. Our analysis is then employed to measure the persistence
length of ds-DNA tethers of three different lengths spanning two orders of
magnitude. The effective persistence length of such tethers is shown to
decrease with the contour length, in accordance with previous studies.
