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.

Electron backscatter diffraction (EBSD) has become a common technique for measuring crystallographic orientations at spatial resolutions on the order of tens of nanometers and at angular resolutions <0.1°. In a recent search of EBSD papers using Google Scholar™, 60% were found to address some aspect of deformation. Generally, deformation manifests itself in EBSD measurements by small local misorientations. An increase in the local misorientation is often observed near grain boundaries in deformed microstructures. This may be indicative of dislocation pile-up at the boundaries but could also be due to a loss of orientation precision in the EBSD measurements. When the electron beam is positioned at or near a grain boundary, the diffraction volume contains the crystal lattices from the two grains separated by the boundary. Thus, the resulting pattern will contain contributions from both lattices. Such mixed patterns can pose some challenge to the EBSD pattern band detection and indexing algorithms. Through analysis of experimental local misorientation data and simulated pattern mixing, this work shows that some of the rise in local misorientation is an artifact due to the mixed patterns at the boundary but that the rise due to physical phenomena is also observed.

Two methods are especially suited for tomographic imaging with histological detail of macroscopic samples that consist of multiple tissue types (bone, muscle, nerve or fat): Light sheet (based) fluorescence microscopy (LSFM) and micro-computed tomography (micro-CT). Micro-CT requires staining with heavy chemical elements (and thus fixation and sometimes dehydration) in order to make soft tissue imageable when measured alongside denser structures. LSMF requires fixation, decalcification, dehydration, clearing and staining with a fluorescent dye. The specimen preparation of both imaging methods is prone to shrinkage, which is often not mentioned, let alone quantified. In this paper the presence and degree of shrinkage are quantitatively identified for the selected preparation methods/stains. LSFM delivers a volume shrinkage of 17% for bone, 56% for muscle and 62% for brain tissue. The three most popular micro-CT stains (phosphotungstic acid, iodine with potassium iodide, and iodine in absolute ethanol) deliver a volume shrinkage ranging from 10 to 56% for muscle and 27–66% for brain, while bone does not shrink in micro-CT preparation.

The purpose of this study was to evaluate the ability of two dentin adhesive systems to induce remineralization in the bonded dentin interface after in vitro thermo-cycling. Dentin surfaces were treated with two different adhesive approaches: (1) 37% phosphoric acid (PA) plus an “etch-and-rinse” dentin adhesive (single bond, SB) (PA+SB) or (2) application of a “self-etch” dentin adhesive (Clearfil SE bond, SEB). Three groups were established: (i) 24 h or (ii) 3 m storage, and (iii) specimens submitted to thermal cycling (100,000 cy/5 and 55ºC). Atomic force microscopy imaging/nanoindentation, Raman spectroscopy/cluster analysis with dye-assisted confocal laser scanning microscopy (CLSM) evaluation and Masson’s trichrome staining assessments were implemented for characterization. Thermo-cycling increased nanohardness in PA+SB at the hybrid layer (HL) and in SEB at the bottom of the HL (BHL). Young’s modulus increased at both the HL and BHL in SEB and at the HL in PA+SB, after thermal stress. Cluster analysis demonstrated an augmentation of the mineral–matrix ratio in thermo-cycled specimens. CLSM showed a decrease of both micropermeability and nanoleakage after thermo-cycling in PA+SB, and were completely absent in SEB. Trichrome staining reflected a scarce demineralized front in PA+SB after thermo-cycling and total remineralization in SEB.

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.

Bacterial endospores are resistant to many environmental factors from temperature extremes to ultraviolet irradiation and are generally more difficult to inactivate or kill than vegetative bacterial cells. It is often considered necessary to treat spores or samples containing spores with chemical fixative solutions for prolonged periods of time (e.g., 1–21 days) to achieve fixation/inactivation to enable electron microscopy (EM) examination outside of containment laboratories. Prolonged exposure to chemical fixatives, however, can alter the ultrastructure of spores for EM analyses. This study was undertaken to determine the minimum amount of time required to inactivate/sterilize and fix spore preparations from several bacterial species using a universal fixative solution for EM that maintains the ultrastructural integrity of the spores. We show that a solution of 4% paraformaldehyde with 1% glutaraldehyde inactivated spore preparations of Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis, and Clostridium perfringens in 30 min, and Bacillus subtilis in 240 min. These results suggest that this fixative solution can be used to inactivate and fix spores from several major groups of bacterial spore formers after 240 min, enabling the fixed preparations to be removed from biocontainment and safely analyzed by EM outside of biocontainment.

A procedure has been developed to follow degradation of energy-dispersive spectroscopy (EDS) X-ray lateral resolution in a variable pressure scanning electron microscope. This procedure is based on evaluation of the EDS profile shape change for different experimental conditions. Some parameters affecting the X-ray resolution in high-vacuum mode have been taken into account. Good agreement between the simulated and experimental EDS profiles shows the reliability of the proposed procedure. A significant improvement in measurement of the EDS profile interfacial distance (DINT) has been achieved.

Aberration-corrected scanning transmission electron microscopy images of the In0.15Ga0.85N active region of a blue light-emitting diode, acquired at ~0.1% of the electron dose known to cause electron beam damage, show no lateral compositional fluctuations, but do exhibit one to four atomic plane steps in the active layer’s upper boundary. The area imaged was measured to be 2.9 nm thick using position averaged convergent beam electron diffraction, ensuring the sample was thin enough to capture compositional variation if it was present. A focused ion beam prepared sample with a very large thin area provides the possibility to directly observe large fluctuations in the active layer thickness that constrict the active layer at an average lateral length scale of 430 nm.

A new in situ Scanning Electron Microscope-Focused Ion Beam-based method to study porous carbon electrodes involving Pt filling of pores from gaseous precursors has been demonstrated to show drastically improved image contrast between the carbon and porous phases when compared with the Si-resin vacuum-impregnation method. Whereas, the latter method offered up to 20% contrast, the new method offers remarkably higher contrast (42%), which enabled fast semi-automated demarcation of carbon boundaries and subsequent binarization of the images with very high fidelity. Tomographic reconstruction of the porous carbon electrode was then obtained from which several morphological parameters were quantified. The porosity was found to be 72±2%. The axial and radial tortuosites were 1.45±0.04 and 1.43±0.04, respectively. Pore size, which is defined to be the distance from the medial axis of the pore to the nearest solid boundary, was quantified. Average pore size determined from the pore size distribution was 90 nm and the corresponding 1 sigma ranges from 45 to 134 nm. Surface-to-volume ratio of the carbon phase was 46.5 µm−1. The ratio of total surface area to the total volume of electrode including pores (i.e., specific surface area) was 13 µm−1.

This study evaluated the influence of fluoride mouth rinses and repolishing on the superficial morphology and color stability of nanofilled resin. About 150 specimens were prepared and polished using aluminum oxide discs for 15 s with a pressure of 2 kg. The experimental groups were divided according to the immersion medium (artificial saliva, 0.5% sodium fluoride, Fluordent Reach, Oral B, Fluorgard) and repolishing procedure (without and with). The specimens were continuously immersed for 1 week. Thereafter, half of each sample was repolished. A color reading was performed after 24 h of immersion in the artificial saliva baseline, after continuous immersion, and after repolishing. The superficial morphology was examined using scanning electron microscopy (SEM) in a qualitative way. Color change (∆E) data were submitted to a mixed analysis of variance using a Shapiro–Wilk test (p>0.05 for the different immersion media) and Sidak’s test (p<0.05 for the differences between groups). In the interaction between the repolishing and the immersion media, Fluorgard showed a statistical difference between the ∆E values with and without repolishing (p<0.0001). On the SEM observations, both Fluordent Reach and Fluorgard caused degradation of the superficial resinous matrix of the composite after continuous immersion. This matrix was removed after repolishing.

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.

Macrophages are widely distributed immune system cells with essential functions in tissue homeostasis, apoptotic cell clearance, and first defense in infections. A distinguishing feature of activated macrophages participating in different situations such as inflammatory and metabolic diseases is the presence of increased numbers of lipid-rich organelles, termed lipid bodies (LBs) or lipid droplets, in their cytoplasm. LBs are considered structural markers of activated macrophages and are involved in different functions such as lipid metabolism, intracellular trafficking, and synthesis of inflammatory mediators. In this review, we revisit the distinct morphology of LB organelles actively formed within macrophages in response to infections and cell clearance, taking into account new insights provided by ultrastructural studies. We also discuss the LB interactions within macrophages, revealed by transmission electron microscopy, with a focus on the remarkable LB–phagosome association and discuss potential links between structural aspects and function.

This study reports physical and chemical changes that occur at early dentin remineralization stages. Extracted human third molars were sectioned to obtain dentin discs. After polishing the dentin surfaces, three groups were established: (1) untreated dentin (UD), (2) 37% phosphoric acid application for 15 s (partially demineralized dentin—PDD), and (3) 10% phosphoric acid for 12 h at 25° C (totally demineralized dentin—TDD). Five different remineralizing solutions were used: chlorhexidine (CHX), artificial saliva (AS), phosphate solution (PS), ZnCl2, and ZnO. Wettability (contact angle), ζ potential and Raman spectroscopy analysis were determined on dentin surfaces. Demineralization of dentin resulted in a higher contact angle. Wettability decreased after immersion in all solutions. ζ potential analysis showed dissimilar performance ranging from −6.21 mV (TDD + AS) up to 3.02 mV (PDD + PS). Raman analysis showed an increase in mineral components after immersing the dentin specimens, in terms of crystallinity, mineral content, and concentration. This confirmed the optimal incorporation and deposition of mineral on dentin collagen. Organic content reflected scarce changes, except in TDD that appeared partially denatured. Pyridinium, as an expression of cross-linking, appeared in all spectra except in specimens immersed in PS.

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.