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Join us in the Taylor Science Center, Room G041 Exploring the dynamic 3D world inside live bacterial cells The crowded bacterial cytoplasm is comprised of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial Genetically Encoded Multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (-3240 to +2700 e) in live Escherichia coli cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, our collaborators developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears sub-diffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent sub-diffusion arises from geometrical confinement within the nucleoid and by the cell boundary.   Originally from Costa Rica, Diana dual-majored in Physics and Electrical Engineering at the University of Costa Rica, where she focused on condensed matter research. She then moved to the US for grad school where she became excited about biophysics and living systems. She received her PhD in Physics from Princeton University, studying the dynamic 3D world inside living bacterial cells using advanced optical microscopy. She is a postdoctoral researcher at Yale University’s Yan Laboratory where Diana is excited to continue learning about bacterial systems, at the larger scale of biofilms.