cells*

The bacterial cytoplasm is an extremely crowded and polydisperse environment and this leads to unexpected anomalous diffusion. We use Genetically Encoded Multimeric nanoparticles (GEMs) to probe the microrheology of the Escherichia coli cytoplasm. We reconstruct three-dimensional trajectories from optical microscopy images obtained with a custom-built biplane microscope. The use of different sized GEM particles enables us to explore the diffusion of objects ranging in size from 20 to 50 nm, similar in scale to ribosomes and other macromolecular complexes in the cell. We observe that for larger particles, the motion is confined to cylindrical shells around the nucleoid, with several diffusion regimes. We also vary the total charge of the fluorescent proteins from -18 e to +22 e and investigate the effect on diffusion. Using small molecule drug treatments, we show progress towards understanding the effects of the nucleoid and cell metabolic state on the 3D diffusion of particles inside bacterial cells.