This study asked how B cell receptors (BCRs) are arranged on resting B cells and what triggers activation. Using DNA-PAINT super-resolution microscopy, the authors found most BCRs exist as monomers, dimers or loose clusters, and built monodisperse model antigens of controlled affinity and valency on a Holliday-junction nanoscaffold. They show antigen binding alone is insufficient to activate the BCR; activation depends on antigen size, rigidity, affinity and avidity — an "antigen footprint" model. Flow Induced Dispersion Analysis (FIDA) was used in a supporting role to confirm the nanoscaffold antigens were monodisperse and to measure their hydrodynamic radii, and supported an antibody affinity estimate. It is an example of using FIDA to confirm monodispersity and measure the size of engineered protein/antigen constructs in solution.