Combining the biological specificity of fluorescence microscopy with topographical features revealed by atomic force microscopy (AFM) provides new insights into cell biology. However, the lack of systematic alignment capabilities especially in scanning-tip AFM has limited the combined application approach as AFM drift leads to increasing image mismatch over time. We present an alignment correction method using the cantilever tip as a reference landmark. Since the precise tip position is known in both the fluorescence and AFM images, exact re-alignment becomes possible. We used beads to demonstrate the validity of the method in a complex artificial sample. We then extended this method to biological samples to depict membrane structures in fixed and living human fibroblasts. We were able to map nanoscale membrane structures, such as clathrin-coated pits, to their respective fluorescent spots. Reliable alignment between fluorescence signals and topographic structures opens possibilities to assess key biological processes at the cell surface such as endocytosis and exocytosis.

Natural organic matter (NOM) generically refers to organic substances found in soils, waters, and sediments. It is the brown-to-black, heterogeneous organic material produced through the diagenetic alteration of plant tissue and microbial biomass via a myriad of biotic and abiotic reactions. Since NOM is the primary source of organic carbon in the earth’s surficial environment, understanding the processes by which NOM is produced is integral to understanding carbon sequestration, contaminant fate and transport, and other earth surface processes. NOM samples (HA0) consist of nonamphiphilic (HA1), lipid-like (L0 and L1), and strongly amphiphilic (HA2) components. Here we present the structure and morphology of self-assembled NOM components based on scanning electron microscopy (SEM), atomic force microscopy (AFM), and electrostatic force microscopy (EFM) characterizations. Effects of surface charge and hydrophobicity/hydrophilicity of the amphiphile on the interaction and resulting structures were investigated using SEM, AFM, and EFM. Data shows that the component’s amphiphilic nature plays a key role in the formation of NOM. SEM data show that aggregates form while AFM/EFM analysis verifies the existence of hydrophobic/hydrophilic moieties in different fractions of HA0. Subsequently, the amphiphilic nature of HA2 will have a substantial effect on interfacial interactions and subsequent self-assembly of HA0’s components.

Chemical nano-tomography of microbial cells in their natural, hydrated state provides direct evidence of metabolic and chemical processes. Cells of the nitrate-reducing Acidovorax sp. strain BoFeN1 were cultured in the presence of ferrous iron. Bacterial reduction of nitrate causes precipitation of Fe(III)-(oxyhydr)oxides in the periplasm and in direct vicinity of the cells. Nanoliter aliquots of cell-suspension were injected into custom-designed sample holders wherein polyimide membranes collapse around the cells by capillary forces. The immobilized, hydrated cells were analyzed by synchrotron-based scanning transmission X-ray microscopy in combination with angle-scan tomography. This approach provides three-dimensional (3D) maps of the chemical species in the sample by employing their intrinsic near-edge X-ray absorption properties. The cells were scanned through the focus of a monochromatic soft X-ray beam at different, chemically specific X-ray energies to acquire projection images of their corresponding X-ray absorbance. Based on these images, chemical composition maps were then calculated. Acquiring projections at different tilt angles allowed for 3D reconstruction of the chemical composition. Our approach allows for 3D chemical mapping of hydrated samples and thus provides direct evidence for the localization of metabolic and chemical processes in situ.

Antibiotic resistance is a major risk to human health, and to provide valuable insights into mechanisms of resistance, innovative methods are needed to examine the cellular responses to antibiotic treatment. Focused ion beam tomography is proposed to image and assess the detailed three-dimensional (3D) ultrastructure of single bacterial cells. By iteratively removing slices of thickness in the order of 10 nm, high magnification 2D images can be acquired by scanning electron microscopy at single-digit nanometer resolution. In this study, Klebsiella pneumoniae was treated with polymyxin B, and 3D models of both cell envelope and cytoplasm regions containing the nucleoid and ribosomes were reconstructed. The 3D volume containing the nucleoid and ribosomes was significantly smaller, and the cell length along the longitudinal axis was extended by 40% in the treated cells, implying stress responses to the drug treatment. More than a 200% increase in protrusions per unit surface area on the cell envelope was observed in the curvature analysis after treatment. Experiments by conventional transmission electron microscopy and atomic force microscopy were also performed, followed by comparison and discussions. In conclusion, the proposed 3D imaging method and associated analysis provide a unique tool for the assessment of antibiotic effects on multidrug-resistant bacteria at nanometer resolution.

In this study, a combined tilt- and focal series is proposed as a new recording scheme for high-angle annular dark-field scanning transmission electron microscopy (STEM) tomography. Three-dimensional (3D) data were acquired by mechanically tilting the specimen, and recording a through-focal series at each tilt direction. The sample was a whole-mount macrophage cell with embedded gold nanoparticles. The tilt–focal algebraic reconstruction technique (TF-ART) is introduced as a new algorithm to reconstruct tomograms from such combined tilt- and focal series. The feasibility of TF-ART was demonstrated by 3D reconstruction of the experimental 3D data. The results were compared with a conventional STEM tilt series of a similar sample. The combined tilt- and focal series led to smaller “missing wedge” artifacts, and a higher axial resolution than obtained for the STEM tilt series, thus improving on one of the main issues of tilt series-based electron tomography.

Polarization microscopy was used for evaluation of kinetics of diclofenac sorption in three different wound dressing materials. The sorption kinetics can be evaluated by radii change and intensity of the light traveling through the fiber. The most frequently used host materials for drugs in wound dressings are alginate, polyesters such as polyethylene terephthalate, and viscose. We studied sorption of diclofenac as an example drug. Effective, but rather simple in vitro simulation of diclofenac sorption gives insight into the applicability of the mentioned materials for development of wound healing materials.

Alkali pretreatment is a promising pretreatment technology that can effectively deconstruct plant cell walls to enhance sugar release performance. In this study, multi-scale visualization of dynamic changes in poplar cell walls during sodium hydroxide pretreatment (2% w/v, 121°C) was carried out by light microscopy (LM), confocal Raman microscopy (CRM) and atomic force microscopy (AFM). LM observations indicated that swelling occurred primarily in the secondary wall (S) but alkali had little effect on the cell corner middle lamella (CCML). Correspondingly, there was a preferential delignification in the S at the beginning of pretreatment, while the level of delignification in CCML (~88%) was higher than that in the S (~83%) for the whole process revealed by Raman spectra. It also suggested that prolonging residence time to 180 min would not remove lignin completely but cause rapid loss of carbohydrates, which was further visualized by Raman spectroscopy images. Furthermore, AFM measurements illustrated that pretreatment with alkali exposed the embedded microfibrils from noncellulosic polymers clearly, enlarged the diameter of microfibrils, and decreased the surface porosity. These results suggested that there was a synergistic mechanism of lignocellulose deconstruction regarding cell wall swelling, main components dissolution, and microfibril morphological changes that occurred during alkali pretreatment.

In situ microanalysis of solid samples is often performed using secondary ion mass spectrometry (SIMS) with a submicron ion probe. The destructive nature of the method makes it mandatory to prevent information loss by using instruments combining efficient collection of secondary ions and a mass spectrometer with parallel detection capabilities. The NanoSIMS meets those requirements with a magnetic spectrometer but its mass selectivity has to be improved for accessing opportunities expected from polyatomic secondary ions. We show here that it is possible to perform D/H ratio measurement images using 12CD−/12CH−, 16OD−/16OH−, or 12C2D−/12C2H− ratios. These polyatomic species allow simultaneous recording of D/H ratios and isotopic compositions of heavier elements like 15N/14N (via 12C15N−/12C14N−) and they provide a powerful tool to select the phase of interest (e.g., mineral versus organics). We present high mass resolution spectra and an example of isotopic imaging where D/H ratios were obtained via the 12C2D−/12C2H− ratio with 12C2D− free from neighboring mass interferences. Using an advanced mass resolution protocol, a “conventional” mass resolving power of 25,000 can be achieved. Those results open many perspectives for isotopic imaging at a fine scale in biology, material science, geochemistry, and cosmochemistry.

Dynamic impedance spectroscopy, designed for measuring nonstationary systems, was used in combination with scanning probe microscopy. Using this approach, impedance mapping could be carried-out simultaneously with topography scanning. Therefore, correlation of electrical properties with particular phases of an examined sample was possible. The sample used in this study was spheroidal graphite cast iron with clearly defined phases having significantly different properties. Additionally, impedance-force curves were made at graphite precipitation and ferrite matrix to illustrate the relation between impedance and the force applied to a probe.

The surface properties of hydroxyapatite, including electric charge, can influence the biological response, tissue compatibility, and adhesion of biological cells and biomolecules. Results reported here help in understanding this influence by creating charged domains on hydroxyapatite thin films deposited on silicon using electron beam irradiation and investigating their shape, properties, and carbon contamination for different doses of incident injected charge by two methods. Photoluminescence laser scanning microscopy was used to image electrostatic charge trapped at pre-existing and irradiation-induced defects within these domains, while phase imaging in atomic force microscopy was used to image the carbon contamination. Scanning Auger electron spectroscopy and Kelvin probe force microscopy were used as a reference for the atomic force microscopy phase contrast and photoluminescence laser scanning microscopy measurements. Our experiment shows that by combining the two imaging techniques the effects of trapped charge and carbon contamination can be separated. Such separation yields new possibilities for advancing the current understanding of how surface charge influences mediation of cellular and protein interactions in biomaterials.

Mimetite, Pb5(AsO4)3Cl, is a ubiquitous mineral that is of interest in various fields such as ore mineralogy, environmental studies, and minerals engineering. Mimetite generally forms hexagonal prisms and barrel-shaped crystals, but its fibrous form is quite an uncommon one. Here we attempt to present its worldwide occurrences on the basis of a literature review. We then present a study on fibrous mimetite from one of its historical locations based on the petrographic microscope, SEM-SE and EDX, and Raman spectroscopy.

The analytical performance of high-resolution scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX) for accurate determination of the size, size distribution, qualitative elemental analysis of nanoparticles (NPs) was systematically investigated. It is demonstrated how powerful high-resolution SEM is by using both mono- and bi-modal distributions of SiO2 airborne NPs collected on appropriate substrates after their generation from colloidal suspension. The transmission mode of the SEM (TSEM) is systematically employed for NPs prepared on thin film substrates such as transmission electron microscopy grids. Measurements in the transmission mode were performed by using a “single-unit” TSEM transmission setup as manufactured and patented by Zeiss. This alternative to the “conventional” STEM detector consists of a special sample holder that is used in conjunction with the in-place Everhart–Thornley detector. In addition, the EDX capabilities for imaging NPs, highlighting the promising potential with respect to exploitation of the sensitivity of the new large area silicon drift detector energy dispersive X-ray spectrometers were also investigated. The work was carried out in the frame of a large prenormative VAMAS (Versailles Project on Advanced Materials and Standards) project, dedicated to finding appropriate methods and procedures for traceable characterization of NP size and size distribution.

Helium accumulation negatively impacts structural materials used in neutron-irradiated environments, such as fission and fusion reactors. Next-generation fission and fusion reactors will require structural materials, such as steels, that are resistant to large neutron doses yet see service temperatures in the range most affected by helium embrittlement. Previous work has indicated the difficulty of experimentally differentiating nanometer-sized cavities such as helium bubbles from the Ti–Y–O rich nanoclusters (NCs) in radiation-tolerant nanostructured ferritic alloys (NFAs). Because the NCs are expected to sequester helium away from grain boundaries and reduce embrittlement, experimental methods to study simultaneously the NC and bubble populations are needed. In this study, aberration-corrected scanning transmission electron microscopy (STEM) results combining high-collection-efficiency X-ray spectrum images (SIs), multivariate statistical analysis (MVSA), and Fresnel-contrast bright-field STEM imaging, have been used for such a purpose. Fresnel-contrast imaging, with careful attention to TEM-STEM reciprocity, differentiates bubbles from NCs. MVSA of X-ray SIs unambiguously identifies NCs. Therefore, combined Fresnel-contrast STEM and X-ray SI is an effective STEM-based method to characterize helium-bearing NFAs.

The development of effective new tools for structural characterization of disordered materials and systems is becoming increasingly important as such tools provide the key to understanding, and ultimately controlling, their properties. The relatively novel technique of correlograph analysis (i.e., the approach of calculating angular autocorrelations within diffraction patterns) promises unique advantages for probing the local symmetries of disordered structures. Because correlograph analysis examines a component of the high-order four-body correlation function, it is more sensitive to medium-range ordering than conventional diffraction methods. As a follow-up of our previous publication, where we studied thin samples of sputtered amorphous silicon, we describe here the practical experimental method and common systematic errors of electron correlograph analysis. Using both experimental data and numerical simulations, we demonstrate that reliable structural information about the sample can only be extracted from the mean correlograph averaged over a sufficient number of individual results.

A dual-beam focused ion beam microscope equipped with a nanomanipulator was used to fabricate slices from within individual hematite (α-Fe2O3) pseudocubes with selected orientations with respect to the original pseudocubes. Transmission electron microanalysis through selected area electron diffraction enabled assignment of each thin section to a particular zone of the hematite lattice. While the pseudocubes are composed of numerous crystallites, 25–50 nm in size, they are not simply polycrystalline particles. Electron diffraction of thin sections showed that while the pseudocubic hematite particles are composed of numerous coherent domains, the individual thin sections display a net crystallographic orientation to the underlying hematite lattice. Quantitative analysis of the lattice misorientation between coherent domains was calculated from the azimuthal spread of electron diffraction peaks and is consistent with a structure that contains small-angle grain boundaries. Based upon this analysis, we conclude that the pseudocubic hematite particles are mosaic crystals, composed of highly oriented coherent domains.