Decoding the molecular interplay of endogenous CD20 and Rituximab with fast volumetric nanoscopy

Elucidating the interaction between membrane proteins and antibodies requires fast whole-cell imaging at high spatiotemporal resolution. Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but suffers from limited spatial resolution. DNA-PAINT achieves molecular resolution but is practically restricted to two-dimensional imaging due to long acquisition times. Here, we introduce two-dye imager (TDI) probes, manifesting negligible background and amplified fluorescence signal upon transient binding, enabling ∼15-fold faster imaging. Using a combination of TDI-DNA-PAINT and LLS microscopy on B cells, we reveal the oligomeric states and interaction of endogenous CD20 with the therapeutic monoclonal antibody rituximab (RTX), unperturbed by surface effects. Our results demonstrate that B cells become polarized, and microvilli stabilized by RTX binding. These findings, we believe, will aid rational design of improved immunotherapies targeting tumor-associated antigens. ### Competing Interest Statement The authors have declared no competing interest.