Streptavidin-functionalized gold nanoparticles (a; with a PEG shell thickness of 5 nm) are linked to biotinylated lipids (linker length of ∼5 nm) in the SLB and label-free monitored using surface-enhanced ellipsometric contrast (SEEC) imaging. (b,c) give a decomposition of a representative trajectory (30 nm gold nanoparticle; 15 μl min−1 flow rate) into its component in flow direction (x-axis) and perpendicular to the flow (y-axis). Due to the flow, the x-component is dominated by a directed movement (indicated by its linear increase with time), allowing to extract the induced velocity vx, while the y-component remains (due to absence of shear force in this direction) fully random and allows to extract the linker diffusion coefficient Dy (see Supplementary Note 3 for details). Combining both information yield the hydrodynamic shear force Fs acting on the particular nanoparticle. Histograms (d) of Fs (after normalization to the flow rate) exhibit a peak at 1.60 fN min μl−1 for 30 nm (blue), at 4.05 fN min μl−1 for 50 nm and at 10.83 fN min μl−1 for 105 nm gold nanoparticles (hydrodynamic radius). These calibration measurements allowed to fit equation (5) (e), which is required to convert distributions of the hydrodynamic force into size distributions. The solid line in e gives the result of a weighted least squares fit that also takes the standard deviation of the Fs distribution (error bars) into account and yields λ=24.4 nm and Aη=1 fN min μl−1.