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We have developed an automated single-molecule imaging system for live-cell analysis based on robotics and artificial intelligence-assisted microscopy. All significant procedures, i.e., searching for cells suitable for observation, detecting in-focus positions, and performing image acquisition and single-molecule tracking, are fully automated. Highly accurate, efficient, and reproducible single-molecule imaging experiments of receptor molecules on the plasma membrane of living cells can be performed. By narrowing the field of view to the size of a single cell, the excitation light is made to be uniform, allowing quantitative measurements of the diffusion and cluster formation of fluorescently labeled receptors. By developing a technology that automatically focuses on membrane receptors at high speed, high-throughput singlemolecule measurements that can observe about 400 fields of view per hour are achieved. We applied this technique to the single-molecule imaging analysis of epidermal growth factor receptors (EGFR). Changes in the lateral diffusion and cluster formation of EGFR on the membrane in response to EGF under various drug treatments, including tyrosine kinase inhibitors (TKIs), were clearly detected in individual cells. The spatiotemporal dynamics of EGFR upon its activation was characterized to determine several diffusion parameters and pharmacological parameters, including the number of diffusion states, diffusion coefficients, oligomer sizes, half-maximal effective concentration, and half-maximal inhibition concentration, showing a powerful potential for discovering novel therapeutics. Therefore, automated single-molecule imaging for systematic cell signaling analysis is feasible and is expected to extensively contribute to biological and pharmacological research.
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