Imaging amyloid fibers at the nanoscale: Method development and applications for hybrid materials and biomedicine

Abstract

In the last decades, advanced imaging techniques have improved our ability to analyze biological systems at the nanoscale, enabling the observation of structural and molecular components. Different imaging tools are specialized in the characterization of a specific aspect of the sample and, when they are combined, complementary information is obtained providing a more comprehensive understanding of the system. This thesis focuses on the application of (super-resolution) fluorescence microscopy in combination with atomic force microscopy (AFM) for revealing specific chemical information in a high-resolution topography map. Particularly, correlative microscopy is applied to the characterization of amyloid fibers, which are misfolded protein aggregates with interest in nanomaterials research and biomedicine. This manuscript is organized in seven chapters. Chapter 1 introduces the imaging techniques used in the thesis. It also gives a general overview on amyloid fibers, their application as hybrid materials, their importance in biomedicine for being involved in different diseases, and the phototherapeutic approaches available to treat them. In Chapter 2, the general materials and methods used during the thesis are explained. Chapter 3 provides a detailed discussion about technical aspects of correlative super-resolution fluorescence microscopy and AFM such as sample preparation, data analysis and image alignment. Furthermore, the advantage of using AFM as a “ground truth” to evaluate different aspects of super-resolution techniques, such as labeling or image reconstruction, is highlighted. In Chapter 4, the methodology developed in Chapter 3 is applied to evaluate the functionalization of amyloid fibers with quantum dots or organic fluorophores. Thus, correlative microscopy is presented as a useful technique for characterizing luminescent hybrid materials at the nanoscale. In the context of biomedicine, amyloid aggregates are important for being involved in different diseases (e.g. Alzheimer or Parkinson). Photochemical strategies to degrade amyloid structures are becoming an interesting alternative. In this thesis, a thioflavin T (ThT) derivative (ROS-ThT), which is able to target pathogenic aggregates in the presence of functional proteins, is used to study photodamage effects on amyloid fibers. In addition to fluorescence, this photocatalyst or photosensitizer produces singlet oxygen Abstract upon blue light exposure, affecting amyloid structures through oxidation. The purpose of Chapter 5 is to select a useful amyloid model to evaluate photodamage at the nanoscale, and therefore different fibers were produced, fibrillated and characterized. In Chapter 6, the selected amyloid model is used to study photodamage induced by ROS-ThT at the single-fiber level through imaging techniques, and complemented by classical biochemical assays. These experiments highlight that the combination of fluorescence microscopy and AFM is useful to probe the heterogeneity of amyloid material and to disentangle the complex dependence between photocatalyst binding/activity and fiber morphology and/or composition. The aim of Chapter 7 is to provide coherence and perspective to the main results of the thesis, as well as an outlook on how advanced microscopy methods may impact the study of amyloids in different fields of research