FIGURE 5.

Effectiveness of selection criteria. (Stage 1) Histogram of the in-plane motion for a collection of tethers that are nominally 1.6 kbp in length. The in-plane motion is defined as in Fig. 3. (Stage 2–4) Histograms for the tethers in Stage 1 that pass successive selection criteria. Stage 2 criterion: the in-plane motion must exceed 10 nm (immobile bead filter). Stage 3 criterion: the in-plane motion of the last 1000 frames must be within 20% of the calculated motion for all frames (adsorption filter). Stage 4 criterion: the in-plane symmetry statistic that measures the ratio of the axes in an ellipsoidal representation of the scatter plot must be <1.2 (malformed tether filter). The arrow in the stage 4 histogram indicates the motion corresponding to 60% of the nominal end-to-end length of the tether (144 nm) that can be used as an additional threshold requirement. Note that as the selection criteria are applied, the distribution of in-plane motion narrows. Microsphere diameter = 560 nm, imaging time = 20–80 s per bead.