Publikationen

Abstract Innovations in liquid-phase electron microscopy (LP-EM) have made it possible to perform experiments at the optimized conditions needed to examine soft matter. The main obstacle is conducting experiments in such a way that electron beam radiation can be used to obtain answers for scientific questions without changing the structure and (bio)chemical processes in the sample due to the influence of the radiation. By overcoming these experimental difficulties at least partially, LP-EM has evolved into a new microscopy method with nanometer spatial resolution and sub-second temporal resolution for analysis of soft matter in materials science and biology. Both experimental design and applications of LP-EM for soft matter materials science and biological research are reviewed, and a perspective of possible future directions is given.

Three-dimensional (3D) printing technologies are widely applied in various industries and research fields and are currently the subject of intensive investigation and development. However, high-performance materials that are suitable for 3D printing are still in short supply, which is a major limitation for 3D printing, particularly for biomedical applications. The physicochemical properties of single constituent materials may not be sufficient to meet the needs of modern biotechnology development and production. To enhance the materials’ performance and broaden their applications, this work designed and tested a series of titanate nanofiller (nanowire and nanotube)-enhanced polycaprolactone (PCL) composites that were 3D-printable and provided superior mechanical properties. By grafting two different functional groups (phenyl- and thiol-terminated ligands), the nanofiller surface showed improved hydrophobicity, which significantly improved their dispersion in the PCL matrix. After characterizing the surface modification, we evaluated the significance of the homogeneity of the ceramic nanofiller in terms of printability, formability, and mechanical strength. Melt electrowriting additive manufacturing was used to fabricate microfibers of PCL and PCL/nanofiller composites. Improved nanofiller dispersion enabled intact and uniform sample morphology, and in contrast, nanofiller aggregation greatly varied the viscosity during the printing process, which could result in poorly printed structures. Importantly, the modified ceramic/PCL composite delivered enhanced and stable mechanical properties, where its Young’s modulus was measured to be 1.67 GPa, which is more than 7 times higher compared to that of pristine PCL (0.22 GPa). Retaining the cell safety properties (comparable to PCL), the concept of enhancing biocompatible polymers with modified nanofillers shows great potential in the field of customized 3D printing for biomedicine.

Liquid phase electron microscopy is a new analytical method that has opened up a rapidly emerging field of research during the past decade. This article discusses this new microscopy modality within the context of imaging eukaryotic cells, bacteria, proteins, viruses, and biomineralization processes. The obtained resolution is typically not a function of the instrument, rather it is limited by the available electron dose within the limit of radiation damage. Therefore, different types of samples are best imaged with different electron microscopy (EM) modalities. The obtained information differs from that acquired with conventional EM as well as cryo-electron microscopy. This article gives an overview of achievements thus far in this area and the unique information that has been obtained. A discussion on potential future developments in the field, and technological advancements required to reach those goals conclude the article.

Quantum dots exhibit unique properties compared to other fluorophores, such as bright fluorescence and lack of photobleaching, resulting in their widespread utilization as fluorescent protein labels in the life sciences. However, their application is restricted to relative quantifications due to lacking knowledge about the labeling efficiency. We here present a strategy for determining the labeling efficiency of quantum dot labeling of HER2 in overexpressing breast cancer cells. Correlative light- and liquid-phase electron microscopy of whole cells was used to convert fluorescence intensities into the underlying molecular densities of the quantum dots. The labeling procedure with small affinity proteins was optimized yielding a maximal labeling efficiency of 83%, which was applicable to the high amount of ∼1.5 × 106 HER2 per cell. With the labeling efficiency known, it is now possible to derive the absolute protein expression levels in the plasma membrane and its variation within a cell and between cells.

The epidermal growth factor receptor HER2 is overexpressed in 20% of breast cancer cases. HER2 is an orphan receptor that is activated ligand-independently by homodimerization. In addition, HER2 is able to heterodimerize with EGFR, HER3, and HER4. Heterodimerization has been proposed as a mechanism of resistance to therapy for HER2 overexpressing breast cancer. Here, a method is presented for the simultaneous detection of individual EGFR and HER2 receptors in the plasma membrane of breast cancer cells via specific labeling with quantum dot nanoparticles (QDs). Correlative fluorescence microscopy and liquid phase electron microscopy were used to analyze the plasma membrane expression levels of both receptors in individual intact cells. Fluorescent single-cell analysis of SKBR3 breast cancer cells dual-labeled for EGFR and HER2 revealed a heterogeneous expression for receptors within both the cell population as well as within individual cells. Subsequent electron microscopy of individual cells allowed the determination of individual receptors label distributions. QD-labeled EGFR was observed with a surface density of (0.5–5) × 101 QDs/µm2, whereas labeled HER2 expression was higher ranging from (2–10) × 102 QDs/µm2. Although most SKBR3 cells expressed low levels of EGFR, an enrichment was observed at large plasma membrane protrusions, and amongst a newly discovered cellular subpopulation termed EGFR-enriched cells.

Nanoparticle superlattice films form at the solid-liquid interface and are important for mesoscale materials, but are notoriously difficult to analyze before they are fully dried. Here, the early stages of nanoparticle assembly were studied at solid-liquid interfaces using liquid-phase electron microscopy. Oleylamine-stabilized gold nanoparticles spontaneously formed thin layers on a silicon nitride (SiN) membrane window of the liquid enclosure. Dense packings of hexagonal symmetry were obtained for the first monolayer independent of the nonpolar solvent type. The second layer, however, exhibited geometries ranging from dense packing in a hexagonal honeycomb structure to quasi-crystalline particle arrangements depending on the dielectric constant of the liquid. The complex structures formed by the weaker interactions in the second particle layer were preserved, while the surface remained immersed in liquid. Fine-tuning the properties of the involved materials can thus be used to control the three-dimensional geometry of a superlattice including quasi-crystals.

Friction of textiles on the human forearm is an important factor in comfort sensations of garments. We built an experiment to measure friction for textiles sliding on the forearm under loading conditions which are characteristic for wearing shirts or jackets. The hair coverage of the participants’ forearm was quantified by image analysis of photographs of the arm in the region of contact. Friction results for five standard textiles suggest to treat hair coverage in two classes. Sweating after physical activity leads to an increase of friction by factors of 2 to 5 for participants with less hairy forearms, while an increase by a factor of 1 to 1.7 only was found for participants with more hairy forearms. We introduce a method of wetting the forearm of study participants in a controlled way with water, which results in similar friction as for the sweating forearm after physical activity. The method allows for efficient studies of the role of skin moisture for friction including varying hair coverage of the skin.

Safe-by-Design aims to reduce uncertainties and/or increase the human health and environmental safety from already early in the innovation process onwards and will thereby contribute to increased innovation efficiency, economic viability, interdisciplinary collaboration, consumers trust and improve sustainability. Since most innovators or designers are neither toxicologists nor risk assessors, considering human health safety aspects within their innovation process may be challenging. This paper provides sets of questions that can help innovators to assess nanospecific human health safety aspects of their product or material along the various stages of the innovation process. Addressing these questions will facilitate innovators to identify which type of information may support decisions on how to address potential human health risks in the innovation process. The identified information on the human health safety aspects can help innovators to decide if further investments in the product or material are beneficial. It may allow them to rank, prioritize and choose safer alternatives early in the innovation process. This may enable innovators to better anticipate on potential safety issues in an early stage, preventing these safety issues to become an innovation killer in a later stage of the innovation process. This approach to identify potential nanospecific human health risks should be considered as complementary to current regulations. The applicability of this approach was evaluated using a few industrial case studies. To determine if the approach is applicable to the innovation of a broader group of nanomaterials and nano-enabled products, more experience within various industrial sectors is needed.

Integration of biophysical stimulation in test systems is established in diverse branches of biomedical sciences including toxicology. This is largely motivated by the need to create novel experimental setups capable of reproducing more closely in vivo physiological conditions. Indeed, we face the need to increase predictive power and experimental output, albeit reducing the use of animals in toxicity testing. In vivo, mechanical stimulation is essential for cellular homeostasis. In vitro, diverse strategies can be used to model this crucial component. The compliance of the extracellular matrix can be tuned by modifying the stiffness or through the deformation of substrates hosting the cells via static or dynamic strain. Moreover, cells can be cultivated under shear stress deriving from the movement of the extracellular fluids. In turn, introduction of physical cues in the cell culture environment modulates differentiation, functional properties, and metabolic competence, thus influencing cellular capability to cope with toxic insults. This review summarizes the state of the art of integration of biophysical stimuli in model systems for toxicity testing, discusses future challenges, and provides perspectives for the further advancement of in vitro cytotoxicity studies.