npSCOPE: A New Multimodal Instrument for In Situ Correlative Analysis of Nanoparticles.

Over the last few decades, nanoparticles have become a key element in a number of scientific and technological fields, spanning from materials science to life sciences. The characterization of nanoparticles or samples containing nanoparticles, in terms of morphology, chemical composition, and other parameters, typically involves investigations with various analytical tools, requiring complex workflows and extending the duration of such studies to several days or even weeks. Here, we report on the development of a new unique in situ correlative instrument, allowing us to answer questions about the shape, size, size distribution, and chemical composition of the nanoparticles using a single probe. Combining various microscopic and analytical capabilities in one single instrument allows a considerable increase in flexibility and a reduction in the duration of such complex investigations. The new instrument is based on focused ion beam microscopy technology using a gas field ion source as a key enabler and combining it with specifically developed secondary ion mass spectrometry and scanning transmission ion microscopy technology. We will present the underlying concept, the instrument and its main components, and proof-of-concept studies performed on this novel instrument. For this purpose, different pure titanium dioxide nanoparticle samples were investigated. Furthermore, the distribution and localization of the nanoparticles in biological model systems were studied. Our results demonstrate the performance and usefulness of the instrument for nanoparticle investigations, paving the way for a number of future applications, in particular, nanotoxicological research.

Salt accumulation and effects within foliage of Tilia × vulgaris trees from the street greenery of Riga, Latvia.

Green infrastructures within sprawling cities provide essential ecosystem services, increasingly undermined by environmental stress. The main objective in this study was to relate the allocation patterns of NaCl contaminants to injury within foliage of lime trees mechanistically and distinguish between the effects of salt and other environmental stressors. Using field material representative of salt contamination levels in the street greenery of Riga, Latvia, the contribution of salt contaminants to structural and ultrastructural injury was analyzed, combining different microscopy techniques. On severely salt-polluted and dystrophic soils, the foliage of street lime trees showed foliar concentrations of Na/Cl up to 13,600/16,750 mg kg-1 but a still balanced nutrient content. The salt contaminants were allocated to all leaf blade tissues and accumulated in priority within mesophyll vacuoles, changing the vacuolar ionic composition at the expense of especially K and Ca. The size of mesophyll cells and vacuoles was increased as a function of NaCl concentration, suggesting impeded transpiration stream. In parallel, the cytoplasm showed degenerative changes, suggesting indirect stress effects. Hence, the lime trees in Riga showed tolerance to the dystrophic environmental conditions enhanced by salt pollution but their leaf physiology appeared directly impacted by the accumulation of contaminants within foliage.

Correlative 3D imaging: CLSM and FIB-SEM tomography using high-pressure frozen, freeze-substituted biological samples.

Correlative light and electron microscopy aims at combining data from different imaging modalities, ideally from the same area of the one sample, in order to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern 3D imaging techniques opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale. Here we present an approach to correlate 3D light microscopy data with volume data from focused ion beam-scanning electron microscopy. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en bloc imaging, is described. It is based on including fluorescent labeling during freeze-substitution, which enables histological context description of the structure of interest by confocal laser scanning microscopy prior to high-resolution electron microscopy. This information can be employed to relocate the respective structure in the electron microscope. This approach is most suitable for targeted small 3D volume correlation and has the potential to extract statistically relevant data of structural details for systems biology.

Freezing continuous-flow self-assembly in a microfluidic device: toward imaging of liposome formation.

A new method is described that combines a microfluidic device for the controlled formation of liposomes with instantaneous immobilization by means of ultrarapid cooling. The microfluidic device is composed of capillaries to hydrodynamically focus a stream of lipids dissolved in 2-propanol by two adjacent aqueous buffer streams before rapidly cooling by propane jet-freezing. The capillary containing the frozen sheath-flow is subsequently separated from the flow-focusing unit and trimmed with cryo-ultramicrotomy for imaging with cryo-scanning electron microscopy (SEM). The emergence of liposomes could be visualized by cryo-SEM without the need for chemical fixation or labeling. We demonstrate that the method is capable of revealing in more detail the formation of nonequilibrium liposomes. Partially and completely formed liposomes were observed at the miscible alcohol-buffer interface. The number density of lipid vesicles varied along the focused interface, and we frequently found clusters of liposomes. Additionally, evidence for the formation of disclike transient intermediates is presented. The method is not limited to studying self-assembly processes only. It can be extended to other biochemical reactions, crystallization processes, and even systematic interfacial mixing studies between different solvents.

Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures.

The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.

Measuring single-nanoparticle wetting properties by freeze-fracture shadow-casting cryo-scanning electron microscopy.

Nanoparticles at fluid interfaces are central to a rapidly increasing range of cutting-edge applications, including drug delivery, uptake through biological membranes, emulsion stabilization and the fabrication of nanocomposites. Understanding nanoscale wetting is a challenging issue, still unresolved for individual nanoparticles, and is essential in designing nanoparticle-building blocks with controlled surface properties. The core information about the structural and thermodynamic properties of particles at fluid interfaces is enclosed in the three-phase contact angle θ. Here we present a novel in situ method, on the basis of freeze-fracture shadow-casting cryo-scanning electron microscopy, that allows the measurement of contact angles of individual nanoparticles with 10 nm diameter, and thus greatly surpasses the current state of the art. We study hydrophilic and hydrophobic, organic and inorganic nanoparticles, demonstrating general applicability to systems of fundamental and applied nanotechnological interest. Significant heterogeneity in the wetting of nanoparticles is observed.