Atomic Force Microscopy of Biological Systems: Quantitative Imaging and Nanomotion Detection

The core of this thesis is the application of different modalities of atomic force microscopy to study living systems. After a brief introduction, quantitative imaging state of the art is presented and the applications for mammalian, plant, bacterial, and yeast cells are reviewed. The same chapter contains research effort about exploring nanomechanical properties of bone cells exposed to conditions that simulate the ones in outer space, in order to investigate the changes in architectural rearrangements of cells during space flight. The next section takes the focus on probing bacterial adhesion sites with an antibody-modified tip, a research important for the understanding of how bacterial cells adhere to the host and establish an infection. Plant cells and their nanomechanical profiles are also covered in the chapter. A large portion of the thesis consists in development of a new nanomechanical sensing technique, and the exploration of its potential applications. The technique is based on the AFM detection method and focuses on transducing small fluctuations that define living systems into a measurable mechanical change in the cantilever. The fact that there is a disproportionate rise in antimicrobial-resistant bacteria and that our lives depend on the new drugs and diagnostic tools to fight them has fueled the first and largest application of the technique – rapid antimicrobial susceptibility resting. Chapter 3 starts with the introduction of the technique, basic principles and guidelines for its use, and continues on potential applications. Proof of principle of the technique is presented, showing experimental results using blind clinical samples of bloodstream infectious agents. A separate section is devoted to investigations concerning slow-growing bacteria and the application of the technique to rapidly determine which antibiotic would best work against them. It then expands on the applications towards detecting metabolic activity of mitochondria and their response to substrates and inhibition. Furthermore, the method has been described as a rapid anti-cancer profiling tool that might pave the way towards a diagnostic platform for personalized medical treatment. The final section of the third chapter contains the current knowledge of the origins of oscillatory movement that governs the nanomotion technique. It presents different hypothesis and results towards exploring them. The thesis ends with a summary of the main ideas and conclusions about the presented work and future perspectives.