Soft matter characterization beyond resolution limit

Abstract

Nature has designed complex machineries and structures that often rely on interactions between nanometer-sized objects. These objects have inspired scientists to rationally design nanomaterials (NMs). Interest in NMs has led to the development of highly impactful applications in fields ranging from biomedicine to coatings. However, their nanometric dimensionality, combined with resolution limits, complicates clear and immediate characterization. This challenge has led to the development of high-resolution microscopy tools and sophisticated analysis algorithms that work in synergy to overcome the limitations of physical resolution. Among these, two award-winning methodologies deserve particular mention: cryogenic electron microscopy (CryoEM) and super-resolution microscopy (SRM). In this thesis, I apply these advanced methods to investigate novel NMs, specifically amyloid peptide nanofibrils (PNFs) and polymeric nanoparticles (NPs). To begin with, I will shows the importance of atomistic investigation of amyloid PNFs. To do so, I combine CryoEM imaging with single particle analysis tools to unravel the structure derived from a novel short self-assemblying peptide. The study discovered that, alongside the common cross-β motif- characteristic in amyloid fibrils- a novel trimeric junction motif is formed. Cooperation of these two motifs elegantly as- sembles with an hexagonal distribution, producing within the fibril a multi-compartment nanotube. This unexpected discovery underscores the importance of structural inves- tigations in nanomaterials, highlighting the potential for tailoring specific applications based on their unique architectures. The characterization analysis in the thesis then shifts to a different type of systems: multicomponent polymeric NPs. Atomistic analysis of these NPs is not yet possible, thus the focus shifted to morphological investigation. Specifically, SRM and electron energy loss spectroscopy were applied to detect the distribution of the distinct chemical moieties within single NPs. Notably, I not only present an overview of a showcase example but rather focus on (semi)-quantitative description. Specifically, I focus on describing composition, size and morphology of small multi-polymeric NPs. The performed image analysis allows critical assessment of the microscopy data removing user-biased image interpretation. Finally, the concluding section of my thesis continues to address polymeric NPs. However, the characterization analysis shifts its focus to the NP surface. The objective of this investigation is to conduct a detailed analysis of the distribution of functional groups. The analysis is conducted from an ensemble average perspective, with the objective of focusing on the distribution of different types of functional groups: total, visible, and accessible. Furthermore, the accessible functional groups are investigated with highresolution microscopy in order to enable particle-to-particle differentiation. In summary, my work presents a fundamental characterization approach for NMs, aiming to unravel fine details that can improve the understanding of their function.