Enhanced Tethered-Particle Motion Analysis Reveals Viscous Effects Sandip Kumar, Carlo Manzo, Chiara Zurla, Suleyman Ucuncuoglu, Laura Finzi, David Dunlap  Biophysical Journal  Volume 106, Issue 2, Pages 399-409 (January 2014) DOI: 10.1016/j.bpj.2013.11.4501 Copyright © 2014 Biophysical Society Terms and Conditions

Figure 1 Representative excursion data. A bead with a radius of 240 nm attached to a DNA tether 2211 bp long was tracked for 400 s. (a–c) xy positions (a), x-positions (b), or y-positions (c) are shown as a function of time. (d) Normalized distribution of the projected distances to the anchor point, ρ, are shown for the same time interval. Biophysical Journal 2014 106, 399-409DOI: (10.1016/j.bpj.2013.11.4501) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 2 Prediction of anchor points as a function of the averaging time. In λ buffer containing 70% glycerol (v/v), a bead with a radius of 160 nm, attached to a DNA tether 1749 bp in length, was tracked for the time intervals indicated. Black dots are the positions of the bead, and the + symbol indicates the anchor point determined by xt,yt (x and y averages). Positions and anchor points are shown for time intervals of 1, 4, 40, or 80 s. An interval of at least 40 s was necessary to accurately predict the anchor point. To see this figure in color, go online. Biophysical Journal 2014 106, 399-409DOI: (10.1016/j.bpj.2013.11.4501) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 3 Mean-square excursion measured for different observation times and viscosities. The mean-square excursion for a bead with a radius of 160 nm attached to a DNA tether 2103 bp long, in λ buffer containing various percentages of glycerol (0, 20, 30, 50, and 70% (v/v)) was calculated using the formula ρt2=((x−xt)2+(y−yt)2)t, for times t = 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56, 5.12, 10.24, 20.48, 40.96, 81.92 s and plotted against log(t). The points corresponding to the same viscosities (percentage of glycerol) are connected using smooth lines. A tethered bead required a longer time to reach the maximum excursion in a higher-viscosity solution. Error bars indicate standard deviations of the time-averaged excursions measured for an ensemble of symmetrically moving tethered beads with closely clustered values. Biophysical Journal 2014 106, 399-409DOI: (10.1016/j.bpj.2013.11.4501) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 4 Calibration of mean-square excursion versus DNA contour length. Mean-square excursion values for two sizes of tethered beads (radii of 160 and 240 nm) in different buffers were calculated using the formula ρt2=((x−xt)2+(y−yt)2)t, with t = 20.48 s. The data, ρ20.48s2, with linear fits are for 240 nm radii beads in λ buffer (<ρ2> = 100.39 × L + 13,383, ⋄), 160-nm-radius beads in λ buffer (<ρ2> = 100.89 × L + 3445, □), 160-nm-radius beads in λ buffer with added Tween (0.5%) (<ρ2> = 91.79 × L + 3594, Δ), and 240-nm-radius beads in TR buffer (<ρ2> = 119.16 × L + 11,475, ○). Error bars indicate standard deviations of the time-averaged excursions measured for an ensemble of symmetrically moving tethered beads with closely clustered values. Biophysical Journal 2014 106, 399-409DOI: (10.1016/j.bpj.2013.11.4501) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 5 Effect of potassium chloride (KCl) concentration on mean-square excursion. The excursions of beads with 240 nm radius attached to 711-, 1052-, or 1267-bp-long DNA tethers, were measured in buffer (10 mM Tris-HCl, pH 7.4, 5% dimethylsulfoxide, 0.1 mM EDTA, 0.2 mM DTT, and 0.1 mg/ml α-casein) supplemented with 10, 50, 100, or 200 mM KCl. The mean-square excursion of beads attached to DNA tethers changed negligibly within the range of KCl used. Error bars indicate standard deviations of the time-averaged excursions measured for an ensemble of symmetrically moving tethered beads with closely clustered values. Biophysical Journal 2014 106, 399-409DOI: (10.1016/j.bpj.2013.11.4501) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 6 Calibration of DNA contour length versus Lx2. Lx2 was determined from the fit of MSDΔt(x) versus Δt for ensembles of tethered beads and plotted as a function of the contour length (L) of the DNA tether. Linear fits to the averaged data were Lx2 = 642.87 × L + 74,416 for beads of radius 240 nm (⋄) or Lx2 = 627.32 × L + 59,670 for beads of radius 160 nm (□). Error bars indicate standard deviations of Lx2 values determined for an ensemble of identically assembled tethered beads. Biophysical Journal 2014 106, 399-409DOI: (10.1016/j.bpj.2013.11.4501) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 7 DcηR versus contour length. DcηR for viscosities 1.006, 2, 3, and 8 cP (0, 20, 30, and 50% glycerol) were calculated and the averages were plotted for tethered beads of 160 nm (□) and 240 nm (⋄) radius as a function of the contour length of the DNA tether. Dc is the diffusion coefficient, η is the viscosity, and R is the radius of the bead. Error bars indicate standard deviations of Dc values determined for ensembles of symmetric, tethered beads. Biophysical Journal 2014 106, 399-409DOI: (10.1016/j.bpj.2013.11.4501) Copyright © 2014 Biophysical Society Terms and Conditions