, where kB is Boltzmann constant, T is temperature, η is the viscosity of water, and r is the estimated effective radius of YOYO-1 that is simplified as a ball in water. When the concentration is known, the only thing needed to predict a bind using our equation is the adsorption cross-section. Please see the Excel file in the supporting information for an example of such calculations with a guessed area. In this report, we report for the first time such a parameter for this example system and probably the first of its kind in all diffusive binding systems. What we were interested in was the initial binding rates of the binding curves, the slopes at time zero, which can be pulled out by fitting a linear line at the initial point of the raw data or with any fitting model e.g. a polynomial function. However, a proper fitting model helps to correct the error caused by concentration fluctuation in the first few seconds when the YOYO-1 solution is just introduced after the buffer washing. It also helps us check the consistency among the curves. As such, the binding data in Fig. was fitted to a Langmuir adsorption model, T + P -> TP, where target T is the number of binding sites in each μm of λDNA, and P is the YOYO-1 probe whose concentration was held constant during the binding. This model yields a single-exponential decay curve for the binding kinetics, 𝑦 = 𝑇 0 -𝑇 0 𝑒 -𝑘𝑡 , where T0 is the available binding sites of 1 μm empty λDNA, k is the binding constant, and t is time. The photobleaching of YOYO-1 was not significant under this laser intensity in a short time window and was convoluted in the binding curve. However, it will be significant for a longer measuring period and thus is included in the fitting to slightly improve the accuracy of the data treatment. For the low probe concentrations, the photobleaching was more significant and the measuring period was relatively small thus a global fitting to hold T0 the same under all conditions is adapted to fit the data. Then the initial binding rates were calculated as T0k from the differential equation of dy/dt at t = 0. These initial binding rates were then used to calculate the binding cross-section using our model 𝑟𝑎𝑡𝑒 = The saturated binding sites are fitted globally for all curves (Fig. ) to be 1030 ± 130 per μm of λDNA, or 3.0 base pair (bp) per binding site, the same value as those reported in the literature , indicating the DNAs are well stretched on the coverslips (Fig. , ~16 μm with 48.5 kbp for a non-broken λDNA). The adsorption cross-section calculated from the initial slope of the fitted curve at time zero decreases over the increase of the probe concentration (Fig. ). We fit the data with an exponential decay function with a saturation baseline. At low concentration limit, the adsorption cross-section is ~3800 nm 2 per μm length of DNA molecules. This value is larger than the area of the DNA ~2000 nm 2 each μm long with a diameter of ~2 nm . The effective size of YOYO-1 is ~1 nm which could have added another 1800 nm 2 area to the effective adsorption cross-section. At the high concentration limit, the adsorption crosssection reduces to ~180 nm 2 per μm DNA, or ~0.2 nm 2 per pair of YOYO-1 and binding site.