Simultaneous Fluorescence and Atomic Force Microscopy to study Mechanically-Induced Bacterial Death in Real Time

Abstract

In the last decades, advanced imaging techniques have improved our ability to analyze biological systems at the micro and nanoscale, and in real time. Microscopy techniques have their own strengths and limitations, so their combination has the potential to provide a more comprehensive understanding of biological processes. This thesis is focused on the development and application of simultaneous fluorescence and atomic force microscopy (AFM) to study mechanically-induced bacterial death. The results reported here provide a quantitative understanding of the mechanical interactions between the AFM tip and bacteria, in the context of emerging mechano-bactericidal nanomaterials. This manuscript is divided into six chapters and one appendix. Chapter 1 provides an overview of the bacterial world and the strategies used over the years to combat the increasing bacterial contamination of surfaces, emphasizing the recent strategy based on mechanical damage. It also describes the microscopy techniques used, highlighting the strengths and weaknesses of each one, and discussing why correlative microscopy is more suitable to study this kind of processes. Chapter 2 describes the general materials and methods applied in this thesis and the software used to analyze experimental data. Chapter 3 provides the groundwork to develop a methodology to successfully combine AFM nanoindentation and fluorescence microscopy simultaneously using fluorescent polymer beads, focusing on the challenges that may arise when simultaneous measurements are performed. In Chapter 4, the methodology was adapted to image bacteria in physiological conditions, and optimal protocols to perform reproducible experiments on living bacteria were found. This optimized methodology in combination with a fluorescent cell membrane integrity marker was successfully applied to quantify the forces needed to rupture the bacterial cell wall. Moreover, a correlation between the forces exerted on bacteria and the kinetics of the fluorescence response is found. Chapter 4 is complemented by Appendix A, which provides the mechanical characterization of the bacterial wall below the rupture point, in order to give a more complete overview of the mechanical properties of the bacterial surface. Chapter 5 explores a different method to assess bacterial viability upon nanoindentation by monitoring the oscillation of the Min system, which reflects bacterial physiology. This method reveals that forces below the breakage point of the cell wall produce a fatigue effect, and provides a quantitative framework to understand low force collisions between bacteria and nanomaterials. These experiments also emphasize the limitation of integrity markers to provide a comprehensive view of bacterial response. The aim of Chapter 6 is to provide coherence and perspective to the main results of the thesis, as well as an outlook on how advanced microscopy methods and future experiments may impact the study of interactions between bacteria and nanotopographical features in the context of mechano-bactericidal nanomaterials