Time-lapse single-molecule imaging revealed spatiotemporal antibody interaction dynamics in situ

Antibody-antigen interactions are primarily characterized by ensemble-averaged affinity measurements outside the cellular environment. However, the highly heterogeneous and dynamic individual antibody behaviors in the cellular environment may influence antibodies to achieve specific functions not well understood at the ensemble level. Here we present single-molecule interaction microscopy (SMIM) as a simple and versatile strategy to explore antibody-antigen interactions in situ. SMIM combines single-molecule localization microscopy with time-lapse imaging, extending single-molecule imaging into sub-minute timescales. Our results indicated that antibodies interact with similar kinetics with both on- and off-targets at the sub-second timescale, while on-target interactions outnumbered off-target interactions at the sub-minute timescale. Furthermore, we observed that immunoglobulin G (IgG) antibodies and antibody fragments (F(ab’)2) display repetitive binding kinetics with their targets on the sub-minute timescale. By extended imaging in the sub-minute timescale, we achieved simultaneous, dual-target, superresolved antibody-antigen interaction maps. SMIM presents an easy-to-implement strategy that enables multiplexed investigation of single antibodies interacting in their native environment with information on the nanoscale topography of both on- and off-targets and temporal dynamics with the on-target.