22While the spatiotemporal structure of the genome is crucial to its biological function, many basic questions 23 remain unanswered on the morphology and segregation of chromosomes. Here, we experimentally show in 24 Escherichia coli that spatial confinement plays a dominant role in determining both the chromosome size 25 and position. In non-dividing cells with lengths up to 10 times normal, single chromosomes are observed 26 to expand more than 4 fold in size, an effect only modestly influenced by deletions of various nucleoid-27 associated proteins. Chromosomes show pronounced internal dynamics but exhibit a robust positioning 28 where single nucleoids reside strictly at mid-cell, while two nucleoids self-organize at ¼ and ¾ cell 29 positions. Molecular dynamics simulations of model chromosomes recapitulate these phenomena and 30 indicate that these observations can be attributed to depletion effects induced by cytosolic crowders. These 31 findings highlight boundary confinement as a key causal factor that needs to be considered for 32 understanding chromosome organization. 33 34 Key words 35 36

Centrifugation is a widespread laboratory technique used to separate mixtures into fractions characterized by a specific size, weight or density. We demonstrate that centrifugation can be also used to separate swimming cells having different motility. To do this we study self-propelled bacteria under the influence of an external centrifugal field. Using dynamic image correlation spectroscopy we measure the spatially resolved motility of bacteria after centrifugation. A significant gradient in swimming-speeds is observed for increasing centrifugal speeds. Our results can be reproduced by a model that treats bacteria as "hot" colloidal particles having a diffusion coefficient that depends on the swimming speed.

Nanophotonics is becoming invaluable for an expanding range of applications, from controlling the spontaneous emission rate and the directionality of quantum emitters, to reducing material requirements of solar cells by an order of magnitude. These effects are highly dependent on the near field of the nanostructure, which constitutes the evanescent fields from propagating and resonant localized modes. Although the interactions between quantum emitters and nanophotonic structures are increasingly well understood theoretically, directly imaging these interactions experimentally remains challenging. Here we demonstrate a photoactivated localization microscopy-based technique to image emitter-nanostructure interactions. For a 75 nm diameter silicon nanowire, we directly observe a confluence of emission rate enhancement, directivity modification and guided mode excitation, with strong interaction at scales up to 13 times the nanowire diameter. Furthermore, through analytical modelling we distinguish the relative contribution of these effects, as well as their dependence on emitter orientation.

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We describe two methods for high-resolution fluorescence imaging of the positioning and mobility of E. coli chemoreceptors fused to photoconvertible fluorescent proteins. Chemoreceptors such as Tar and Tsr are transmembrane proteins expressed at high levels (thousands of copies per cell). Together with their cognate cytosolic signaling proteins, they form clusters on the plasma membrane. Theoretical models imply that the size of these clusters is an important parameter for signaling, and recent PALM imaging has revealed a broad distribution of cluster sizes. We describe experimental setups and protocols for PALM imaging in fixed cells with ~10 nm spatial precision, which allows analysis of cluster-size distributions, and localized-photoactivation single-particle tracking (LPA-SPT) in live cells at ~10 ms temporal resolution, which allows for analysis of cluster mobility.