  Biological nanoparticles (BNPs) are of high interest due to their key role in
various biological processes and use as biomarkers. BNP size and molecular
composition are decisive for their functions, but simultaneous determination of
both properties with high accuracy remains challenging, which is a severe
limitation. Surface-sensitive microscopy allows one to precisely determine
fluorescence or scattering intensity, but not the size of individual BNPs. The
latter is better determined by tracking their random motion in bulk, but the
limited illumination volume for tracking this motion impedes reliable intensity
determination. We here show that attaching BNPs (specifically, vesicles and
functionalized gold NPs) to a supported lipid bilayer, subjecting them to a
hydrodynamic flow, and tracking their motion via surface-sensitive imaging
enable to determine their diffusion coefficients and flow-induced drift
velocities and to accurately quantify both BNP size and emission intensity. For
vesicles, the high accuracy is demonstrated by resolving the expected
radius-squared dependence of their fluorescence intensity.
