Studies of Single-Molecule Dynamics in Microorganisms

Fluorescence microscopy is one of the most extensively used techniques in the life sciences. Considering the non-invasive sample preparation, enabling live-cell compliant imaging, and the specific fluorescence labeling, allowing for a specific visualization of virtually any cellular compound, it is possible to localize even a single molecule in living cells. This makes modern fluorescence microscopy a powerful toolbox. In the recent decades, the development of new, "super-resolution" fluorescence microscopy techniques, which surpass the diffraction limit, revolutionized the field. Single-Molecule Localization Microscopy (SMLM) is a class of super-resolution microscopy methods and it enables resolution of down to tens of nanometers. SMLM methods like Photoactivated Localization Microscopy (PALM), (direct) Stochastic Optical Reconstruction Microscopy ((d)STORM), Ground-State Depletion followed by Individual Molecule Return (GSDIM) and Point Accumulation for Imaging in Nanoscale Topography (PAINT) have allowed to investigate both, the intracellular spatial organization of proteins and to observe their real-time dynamics at the single-molecule level in live cells. The focus of this thesis was the development of novel tools and strategies for live-cell SingleParticle Tracking PALM (sptPALM) imaging and implementing them for biological research. In the first part of this thesis, I describe the development of new Photoconvertible Fluorescent Proteins (pcFPs) which are optimized for sptPALM lowering the phototoxic damage caused by the imaging procedure. Furthermore, we show that we can utilize them together with Photoactivatable Fluorescent Proteins (paFPs) to enable multi-target labeling and read-out in a single color channel, which significantly simplifies the sample preparation and imaging routines as well as data analysis of multi-color PALM imaging of live cells. In parallel to developing new fluorescent proteins, I developed a high throughput data analysis pipeline. I have implemented this pipeline in my second project, described in the second part of this thesis, where I have investigated the protein organization and dynamics of the CRISPR-Cas antiviral defense mechanism of bacteria in vivo at a high spatiotemporal level with the sptPALM approach. I was successful to show the differences in the target search dynamics of the CRISPR effector complexes as well as of single Cas proteins for different target complementarities. I have also first data describing longer-lasting bound-times between effector complex and their potential targets in vivo, for which only in vitro data has been available till today. In summary, this thesis is a significant contribution for both, the advances of current sptPALM imaging methods, as well as for the understanding of the native behavior of CRISPR-Cas systems in vivo

Mathematical models for chemotaxis and their applications in self-organisation phenomena

Chemotaxis is a fundamental guidance mechanism of cells and organisms, responsible for attracting microbes to food, embryonic cells into developing tissues, immune cells to infection sites, animals towards potential mates, and mathematicians into biology. The Patlak-Keller-Segel (PKS) system forms part of the bedrock of mathematical biology, a go-to-choice for modellers and analysts alike. For the former it is simple yet recapitulates numerous phenomena; the latter are attracted to these rich dynamics. Here I review the adoption of PKS systems when explaining self-organisation processes. I consider their foundation, returning to the initial efforts of Patlak and Keller and Segel, and briefly describe their patterning properties. Applications of PKS systems are considered in their diverse areas, including microbiology, development, immunology, cancer, ecology and crime. In each case a historical perspective is provided on the evidence for chemotactic behaviour, followed by a review of modelling efforts; a compendium of the models is included as an Appendix. Finally, a half-serious/half-tongue-in-cheek model is developed to explain how cliques form in academia. Assumptions in which scholars alter their research line according to available problems leads to clustering of academics and the formation of "hot" research topics.Comment: 35 pages, 8 figures, Submitted to Journal of Theoretical Biolog

Liquid-Liquid Phase Separation in an Elastic Network

Living and engineered systems rely on the stable coexistence of two interspersed liquid phases. Yet surface tension drives their complete separation. Here we show that stable droplets of uniform and tuneable size can be produced through arrested phase separation in an elastic matrix. Starting with an elastic polymer network swollen by a solvent mixture, we change the temperature or composition to drive demixing. Droplets nucleate and grow to a stable size that is tuneable by the network cross-linking density, the cooling rate, and the composition of the solvent mixture. We discuss thermodynamic and mechanical constraints on the process. In particular, we show that the threshold for macroscopic phase separation is altered by the elasticity of the polymer network, and we highlight the role of internuclear correlations in determining the droplet size and polydispersity. This phenomenon has potential applications ranging from colloid synthesis and structural colour to phase separation in biological cells.Comment: 6 figure

Roadmap on semiconductor-cell biointerfaces.

This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world

Design Guidelines for Agent Based Model Visualization

In the field of agent-based modeling (ABM), visualizations play an important role in identifying, communicating and understanding important behavior of the modeled phenomenon. However, many modelers tend to create ineffective visualizations of Agent Based Models (ABM) due to lack of experience with visual design. This paper provides ABM visualization design guidelines in order to improve visual design with ABM toolkits. These guidelines will assist the modeler in creating clear and understandable ABM visualizations. We begin by introducing a non-hierarchical categorization of ABM visualizations. This categorization serves as a starting point in the creation of an ABM visualization. We go on to present well-known design techniques in the context of ABM visualization. These techniques are based on Gestalt psychology, semiology of graphics, and scientific visualization. They improve the visualization design by facilitating specific tasks, and providing a common language to critique visualizations through the use of visual variables. Subsequently, we discuss the application of these design techniques to simplify, emphasize and explain an ABM visualization. Finally, we illustrate these guidelines using a simple redesign of a NetLogo ABM visualization. These guidelines can be used to inform the development of design tools that assist users in the creation of ABM visualizations.Visualization, Design, Graphics, Guidelines, Communication, Agent-Based Modeling