Phenotyping by Molecular Mobility: Low-Affinity Reversible Probes Enable Characterizing and Classifying Cells Populations by Single-Molecule Imaging

Cell surface biomolecules, such as membrane receptors, are key drivers of cellular behavior and are therefore inherently different for cell types with different functions and pathogenicity. Here, we present a super-resolution method for functional phenotyping of cell populations based on the single-molecule analysis of membrane receptor mobility. Central to our approach is a library of nanofitin-based low-affinity probes that selectively and reversibly bind to target receptors in live cells. These probes enable the direct detection, counting, and tracking of individual receptors without requiring genetic modification or chemical labeling, allowing the study of endogenous biomolecules in unperturbed cells. Using this strategy, we demonstrate that identical receptors can exhibit distinct mobility patterns across different cell types, reflecting the underlying physiological differences. By integrating these probes with single-molecule point accumulation for imaging in nanoscale topography imaging and tracking, advanced image analysis, and AI-driven classification, we provide a robust workflow for understanding and phenotyping of cells by their receptor mobility.