Figure 3.

Physical models of TFM. (A) Experimental measurements of the FIW for double stranded DNAs 87 to 4000 bp in backbone length. Data were acquired at 1 Hz, and error bars indicate the standard deviation of three independent measurements for each sample. The solid line is the best fit of persistence length to experimental data (P = 48 nm). We observe transient sticking of the DNA end to the surface in less than 1% of traces. (B) The resolution of TFM. Three methods were used to quantify the uncertainty in FIW for DNAs of 87 and 3000 bp. The experimentally determined uncertainties for individual molecules, from 100-frame movies recorded at 2 Hz with illumination powers in the range 1 to 50 mW, are shown as crosses. The intensity plotted is the photon count for a single molecule. Dashed lines are power-law fits as guides for the eye. Equation 12 was used to plot the analytical approximation (solid lines). A simulation, using the parameters of our microscope and the theoretical images of the DNA, generated as described in Materials and Methods, was used to produce the data shown as circles. We observe a good agreement between experiment, simulations, and our analytical expression, with the approximations made in deriving the analytical expression tending to underestimate the noise. For the 3000 bp DNA, deviations can be observed at intensities > 3000 photons, which we attribute to incomplete sampling of the conformations available to the DNA during a single camera frame. The background count in experiments was found to vary linearly with the observed photon count, with 5.3 × 10^-3 background photons per recovered photon for the 87 bp DNA and 9.4 × 10^-3 for the 3000 bp DNA; this was used in our simulations and in the plotting of the analytical approximation. To see this figure in color, go online.