Exploring the dynamic 3D world inside bacteria with advanced optical microscopy

Abstract

The bacterial cytoplasm is extremely crowded and polydisperse, with biomolecules spanning many orders of magnitude in size and electrical charge. In Chapter 2, we study this environment by reconstructing the 3D motion of size and charge-controlled nanoparticles (GEMs) with nanometer-scale resolution in live Escherichia coli cells. GEMs range in size from 20 to 50 nm and in charge from -2160 to 1800 e, similar to important macromolecules. In collaboration with theorists at Stanford, we show that GEMs spatially segregate by size and charge due to cytoplasmic polydispersity and charge interactions with cellular components. Using biplane microscopy for 3D tracking, Monte Carlo simulations, and full-cell colloidal simulations by our collaborators, we find that regardless of charge and particle size, the motion in bacterial cells is mostly normally diffusive. However, this motion appears sub-diffusive due to geometrical confinement to the small cellular volume. We make progress towards understanding the role of chromosome remodeling on particle diffusion in bacterial cells. We show that 50 nm GEMs in exponentially growing cells experience caging, inhibiting the motion of a fraction of the particles. This caging does not occur for smaller GEMs in exponential phase or for 50 nm GEMs in stationary phase cells, suggesting that caging stems from the DNA pore size and active chromosomal re-arrangements. Most of this thesis focuses on freely diffusing particles. However, there is a wealth of interesting phenomena occurring in tethered systems, where particle localization is determined by the anchoring of the molecule, and both the local environment and the tethering action influence dynamics. This is the case for chromosomal loci. In Chapter 4, I conclude by describing steps we have taken towards studying the physics of the bacterial chromosome and propose experiments to study this system using the techniques developed in Chapter 2. In Chapter 3, I present our work rebuilding and optimizing a Stimulated Emission Depletion super resolution microscope. This technique enables sub-diffraction limit imaging by using optical patterning to selectively de-activate fluorescent probes during image acquisition. Although modest super-resolution was achieved, the microscope was never used for biological imaging due to its optical and biological limitations.