X-Ray imaging applied to the characterization of polymer foam's cellular structure and its evolution

Las espumas poliméricas son materiales celulares que poseen una fase sólida continua y otra gaseosa bien discontinua (celda cerrada) o continua (celda abierta). Habitualmente estas estructuras se describen mediante parámetros macroscópicos como la densidad relativa y otros microscópicos como el tamaño de celda o la densidad de celdas. Además, estos materiales poseen características peculiares como anisotropía, orientación de los poros y tortuosidad que les proporcionan propiedades físicas singulares. Convencionalmente el estudio de las espumas poliméricas se realiza mediante el análisis de la estructura celular final obtenida. Ello se debe principalmente a que es complicado detener el proceso de expansión una vez se ha iniciado. Debido a esto los estadios intermedios durante los procesos de espumado no son accesibles, es decir, no se obtiene información acerca de los mecanismos que generan la estructura final. Estos mecanismos físico-químicos fundamentales que gobiernan la generación y evolución de la estructura celular durante el espumado son la nucleación y el crecimiento. Por el contrario, existen otros mecanismos que son responsables de la degeneración de la estructura celular son el drenaje, la coalescencia y el coarsening. Los inconvenientes que existen para abordar el estudio de estos mecanismos durante el proceso de espumado, junto con las peculiaridades de estos sistemas hacen que las técnicas de imagen mediante rayos X sean una herramienta extraordinaria para el estudio in-situ de la evolución de la estructura celular y los mecanismos de espumado. Además, de manera complementaria, la imagen mediante rayos X permite la obtención de tomogramas para el estudio de la estructura celular en el estado final. Incluso es posible llegar más lejos gracias a los últimos desarrollos en tomografía rápida. Esta técnica es capaz de estudiar en 3D la evolución de la estructura celular en el tiempo. Uno de los requisitos esenciales para el estudio de las espumas poliméricas mediante la imagen con rayos X y que condiciona su aplicabilidad es la correcta selección de los componentes y el diseño del equipo de imagen. Esto se debe principalmente a características intrínsecas a las espumas poliméricas: baja absorción de los rayos X, espesores reducidos, estructura de celdas en el rango micrométrico, rápida evolución durante su fabricación y otras peculiaridades morfológicas de su estructura.Departamento de Física de la Materia Condensada, Cristalografía y Minerealogí

Three-dimensional visualization and characterization of polymeric self-assemblies by Transmission Electron Microtomography

Self-assembling structures and their dynamical processes in polymeric systems have been investigated using three-dimensional transmission electron microscopy (3D-TEM). Block copolymers (BCPs) self-assemble into nanoscale periodic structures called microphase-separated structures, a deep understanding of which is important for creating nanomaterials with superior physical properties, such as high-performance membranes with well-defined pore size and high-density data storage media. Because microphase-separated structures have become increasingly complicated with advances in precision polymerization, characterizing these complex morphologies is becoming increasingly difficult. Thus, microscopes capable of obtaining 3D images are required. In this article, we demonstrate that 3D-TEM is an essential tool for studying BCP nanostructures, especially those self-assembled during dynamical processes and under confined conditions.The first example is a dynamical process called order-order transitions (OOTs). Upon changing temperature or pressure or applying an external field, such as a shear flow or electric field, BCP nanostructures transform from one type of structure to another. The OOTs are examined by freezing the specimens in the middle of the OOT and then observing the boundary structures between the preexisting and newly formed nanostructures in three-dimensions. In an OOT between the bicontinuous double gyroid and hexagonally packed cylindrical structures, two different types of epitaxial phase transition paths are found. Interestingly, the paths depend on the direction of the OOT. The second example is BCP self-assemblies under confinement that have been examined by 3D-TEM. A variety of intriguing and very complicated 3D morphologies can be formed even from the BCPs that self-assemble into simple nanostructures, such as lamellar and cylindrical structures in the bulk (in free space).Although 3D-TEM is becoming more frequently used for detailed morphological investigations, it is generally used to study static nanostructures. Although OOTs are dynamical processes, the actual experiment is done in the static state, through a detailed morphological study of a snapshot taken during the OOT. Developing time-dependent nanoscale 3D imaging has become a hot topic. Here, the two main problems preventing the development of in situ electron tomography for polymer materials are addressed. First, the staining protocol often used to enhance contrast for electrons is replaced by a new contrast enhancement based on chemical differences between polymers. In this case, no staining is necessary. Second, a new 3D reconstruction algorithm allows us to obtain a high-contrast, quantitative 3D image from fewer projections than is required for the conventional algorithm to achieve similar contrast, reducing the number of projections and thus the electron beam dose. Combini

Targeted Nucleic Acid Delivery to the Endothelium

The human endothelium extends throughout nearly all tissues in the body, and has an estimated total surface area of up to seven thousand square meters [1]. This expansive monolayer of endothelial cells (ECs) serves as the interface between materials circulating in the bloodstream and the internal tissues of the body. Even in its quiescent state, the endothelium is actively signaling and reacting in order to support the basic functions of the vascular system – transporting oxygen, nutrients, and waste. A closer look reveals that the cells that make up this endothelial lining are diverse in their phenotypes and functions, dependent on the organ or tissue in which they reside. When activated under inflammatory conditions or in other disease states, endothelial cells further differentiate themselves through expression of surface antigens. Being a large, easily accessible surface that interfaces with almost all other tissues in the body, and consisting of distinctly identifiable subcategories as well as some universal characteristics, the endothelium is an attractive therapeutic target. Of particular interest in this dissertation is applying gene therapies to treat or prevent inflammation.We describe a polymeric delivery system for nucleic acids that can be modulated by exchanging polymer end-groups and conjugating cell surface targeting molecules to the delivery vehicle. We show that conjugating EC targeting antibodies to the surface of a cationic poly(amine-co-ester) (PACE) nucleic acid delivery vehicle enhances its transfection efficiency in cultured human ECs. This can enable delivery of gene therapies to ECs, either in vitro as cellular components of an engineered vascular graft, ex vivo in donated human organs for transplantation, or for targeting the endothelium in vivo. In the work presented here we apply the EC-targeted polymeric delivery vehicle to deliver siRNA against IL-15, a cytokine involved the activation of T cells during acute inflammation. Our work with transplant-declined human organs motivated the development of a new digital pathology tool for color-based quantification of histologic specimens, which we have applied to quantify vascular assembly in engineered grafts as well as vascular pathologies in human and animal tissue samples. We demonstrate the benefits of an automated program for color-based detection of pathological features in histologic specimens, in particular in a setting in which large numbers of images are generated. We propose further investigations into the antibody-targeted PACE polyplex delivery platform including a broader exploration of targeting in different cell types in vitro and in vivo. The work described in this dissertation aims to advance the therapeutic potential of targeted nanocarriers for treating pathologies in the endothelium

Assessing cell migration in hydrogels: An overview of relevant materials and methods

Cell migration is essential in numerous living processes, including embryonic development, wound healing, immune responses, and cancer metastasis. From individual cells to collectively migrating epithelial sheets, the locomotion of cells is tightly regulated by multiple structural, chemical, and biological factors. However, the high complexity of this process limits the understanding of the influence of each factor. Recent advances in materials science, tissue engineering, and microtechnology have expanded the toolbox and allowed the development of biomimetic in vitro assays to investigate the mechanisms of cell migration. Particularly, three-dimensional (3D) hydrogels have demonstrated a superior ability to mimic the extracellular environment. They are therefore well suited to studying cell migration in a physiologically relevant and more straightforward manner than in vivo approaches. A myriad of synthetic and naturally derived hydrogels with heterogeneous characteristics and functional properties have been reported. The extensive portfolio of available hydrogels with different mechanical and biological properties can trigger distinct biological responses in cells affecting their locomotion dynamics in 3D. Herein, we describe the most relevant hydrogels and their associated physico-chemical characteristics typically employed to study cell migration, including established cell migration assays and tracking methods. We aim to give the reader insight into existing literature and practical details necessary for performing cell migration studies in 3D environments.publishedVersio

Advances in Focused Ion Beam Tomography for Three-Dimensional Characterization in Materials Science

Over the years, FIB-SEM tomography has become an extremely important technique for the three-dimensional reconstruction of microscopic structures with nanometric resolution. This paper describes in detail the steps required to perform this analysis, from the experimental setup to the data analysis and final reconstruction. To demonstrate the versatility of the technique, a comprehensive list of applications is also summarized, ranging from batteries to shale rocks and even some types of soft materials. Moreover, the continuous technological development, such as the introduction of the latest models of plasma and cryo-FIB, can open the way towards the analysis with this technique of a large class of soft materials, while the introduction of new machine learning and deep learning systems will not only improve the resolution and the quality of the final data, but also expand the degree of automation and efficiency in the dataset handling. These future developments, combined with a technique that is already reliable and widely used in various fields of research, are certain to become a routine tool in electron microscopy and material characterization

Comprehensive study of dynamic curing effect on tablet coating structure

International audienceThe dissolution method is still widely used to determine curing end-points to ensure long-term stability of film coatings. Nevertheless, the process of curing has not yet been fully investigated. For the first time, joint techniques were used to elucidate the mechanisms of dynamic curing over time from ethylcellulose (Aquacoat (R))-based coated tablets. X-ray micro-computed tomography (X mu CT), Near Infrared (NIR), and Raman spectroscopies as well as X-ray microdiffraction were employed as non-destructive techniques to perform direct measurements on tablets. All techniques indicated that after a dynamic curing period of 4 h, reproducible drug release can be achieved and no changes in the microstructure of the coating were any longer detected. X mu CT analysis highlighted the reduced internal porosity, while both NIR and Raman measurements showed that spectral information remained unaltered after further curing. X-ray microdiffraction revealed densification of the coating layer with a decrease in the overall coating thickness of about 10 pm as a result of curing. In addition, coating heterogeneity attributed to cetyl alcohol was observed from microscopic images and Raman analysis. This observation was confirmed by X-ray microdiffraction that showed that crystalline cetyl alcohol melted and spread over the coating surface with curing. Prior to curing, X-ray microdiffraction also revealed the existence of two coating zones differing in crystalline cetyl alcohol and sodium lauryl sulfate concentrations which could be explained by migration of these constituents within the coating layer. Therefore, the use of non-destructive techniques allowed new insights into tablet coating structures and provided precise determination of the curing end-point compared to traditional dissolution testing. This thorough study may open up new possibilities for process and formulation control