or by tracking its actual trajectory in time (e.g. as done in [@Pouget2004] and the present work). Changes in the extent of the motion (which we will call “excursion”) reflect conformational transformations of the tethered molecule. Such changes may be caused by processive walking of RNA polymerase [@Schafer1991; @Yin1994], DNA looping [@Finzi1995; @Broek2006; @Zurla2006; @Zurla2007; @Wong2007; @Vanzi2006; @Beausang2007], DNA hybridization [@Zocchi2003], DNA bending [@Toli2006], Holliday junction formation [@Pouget2004] or RNA translation [@Vanzi2003].

Although TPM is simple in principle, there are a variety of technical challenges that must be addressed for successful implementation. For example, sample preparation can be compromised by multiply-tethered beads, non-specific adsorption, transient sticking events and dissociation of the tether joints [@Pouget2004; @Vanzi2006; @Broek2006; @Blumberg2005; @Nelson2006]. In addition, image analysis of TPM data is complicated by instrumental drift and the stochastic nature of the tethered particle’s motion. Several time scales must be considered, including the total observation time, exposure time, and the intrinsic diffusive time scale of the tethered particle. We will show that quantification of the spatial and temporal resolution of TPM measurements requires an understanding of how particle motion depends on tether length, particle size and other controllable parameters. We focus exclusively on TPM behavior in 