3D single-molecule tracking can determine the subcellular localizations and motion behaviors of individual proteins. Observing a protein's motion in a living cell provides important insights about its interaction with binding partners in its nativ
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3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.