The directional dark-field signal obtained with X-ray grating interferometry yields direction-dependent information about the X-ray scattering taking place inside the examined sample. It allows examination of its morphology without the requirement of resolving the micrometer size structures directly causing the scattering. The local morphology in turn gives rise to macroscopic mechanical properties of the investigated specimen. In this study, we investigate the relation between the biomechanical elasticity (Young’s modulus) and the measured directional dark-field parameters of a well-defined sample made of wood. In our proof-of-principle experiment, we found a correlation between Young’s modulus, the average dark-field signal, and the average dark-field anisotropy. Hence, we are able to show that directional dark-field imaging is a new method to predict mechanical sample properties. As grating interferometry provides absorption, phase-contrast, and dark-field data at the same time, this technique appears promising to combine imaging and mechanical testing in a single testing stage. Therefore, we believe that directional dark-field imaging will have a large impact in the materials science world.

Eukaryotes rely on mitochondrial division to guarantee that each new generation of cells acquires an adequate number of mitochondria. Mitochondrial division has long been thought to occur by binary fission and, more recently, evidence has supported the idea that binary fission is mediated by dynamin-related protein (Drp1) and the endoplasmic reticulum. However, studies to date have depended on fluorescence microscopy and conventional electron microscopy. Here, we utilize whole cell cryo-electron tomography to visualize mitochondrial division in frozen hydrated intact HeLa cells. We observe a large number of relatively small mitochondria protruding from and connected to large mitochondria or mitochondrial networks. Therefore, this study provides evidence that mitochondria divide by budding.

Peyer's patches, macroscopic aggregates of lymphoid follicles present throughout the small intestines of humans and other mammals, are considered the gateway through which luminal dietary antigens and microbes are sampled by the mucosal immune system. The cellular make-up of Peyer's patch lymphoid follicles is not only complex, but highly dynamic, as there are at least four major cell types that are known to migrate in response to antigenic stimulation. In an effort to capture the complexity and dynamic nature of this specialized tissue, here we report the three-dimensional (3D) reconstruction of immunofluorescent-labeled mouse Peyer's patch cryosections. The technology that enabled the stacking and linear blending of serial cryosections was a novel macro for Fiji, the open source image-processing package based on ImageJ. By simultaneously labeling cryosections for surface markers CD45R, CD3, and CD11c, we provide a 3D image as well as quantitative measures of B-cell, T-cell, and dendritic cell populations at steady state and following exposure to the mucosal adjuvant cholera toxin.

Organophosphate pesticides (OPs) like malathion interfere with normal ovarian function resulting in an increased incidence of atresia and granulosa cell apoptosis that plays a consequential role in the loss of ovarian follicles or follicular atresia. The aim of present study was to assess malathion-induced (100 nM) reproductive stress, ultrastructural damage and changes in apoptosis frequency in ovarian granulosa cells of antral follicles. Transmission electron microscopy (TEM) was employed for ultrastructural characterization, oxidative stress was evaluated using thiobarbituric acid reactive substances (TBARS) assay to measure lipid peroxidation, and apoptosis was quantified via flow cytometry. By TEM, apoptosis was identified by the presence of an indented nuclear membrane with blebbing, pyknotic crescent-shaped fragmented nuclei, increased vacuolization, degenerating mitochondria, and lipid droplets. The results indicate a significant increase in malondialdehyde (MDA) level (nmols/g wet tissue) at a 100 nM dose of malathion i.e. 7.57±0.033*, 8.53±0.12*, and 12.87±0.78** at 4, 6, or 8 h, respectively, as compared with controls (6.07±0.033, p<0.01*, p<0.05**) showing a positive correlation between malathion-induced lipid peroxidation and percentage of granulosa cell apoptosis (r=1; p<0.01). The parallel use of these three methods enabled us to determine the role of malathion in inducing apoptosis as a consequence of cytogenetic damage and oxidative stress generated in granulosa cells of antral follicles.

Total internal reflection fluorescence microscopy of fluorescently labeled secretory granules permits monitoring of exocytosis and the preceding granule behavior in one experiment. While observer-dependent evaluation may be sufficient to quantify exocytosis, most of the other information contained in the video files cannot be accessed this way. The present program performs observer-independent detection of exocytosis and tracking of the entire submembrane population of insulin granules. A precondition is the exact localization of the peak of the granule fluorescence. Tracking is based on the peak base radius, peak intensity, and the precrossing itineraries. Robustness of the tracking was shown by simulated tracks of original granule patterns. Mobility in the X–Y dimension is described by the caging diameter which in contrast to the widely used mean square displacement has an inherent time resolution. Observer-independent detection of exocytosis in MIN6 cells labeled with insulin-EGFP is based on the maximal decrease in fluorescence intensity and position of the centroid of the dissipating cloud of released material. Combining the quantification of KCl-induced insulin exocytosis with the analysis of prefusion mobility showed that during the last 3 s pre-exocytotic granules had a smaller caging diameter than control granules and that it increased significantly immediately before fusion.

Scanning electron microscopy (SEM) of scales in six species of the fish genus Channa revealed certain features relevant to taxonomic significance. The location of focus, inter-radial distance and width of circuli, inter-circular space, width of radii, shape and size of lepidonts, etc. were found to be different in different species. The importance of SEM of scales in poorly understood taxonomy and phylogeny of the fish genus Channa is discussed with the help of relevant literature. Further, the role of SEM of fish scales for taxonomic applications is discussed in detail.

The millivolt energy resolution now obtainable in electron energy-loss spectra (EELS) on the latest monochromated scanning transmission electron microscope corresponds, via the uncertainty principle, to a time range of 414 fs (for 10 meV resolution), and a time resolution of 0.138 fs (for energy range of 30 eV). (Thus, the width of an EELS peak is inversely related to the lifetime of an excitation.) This compares favorably with the latest X-ray free electron lasers. The time evolution of a Drude–Lorentz oscillator may be obtained from an EELS using logarithmic deconvolution followed by Kramers–Kronig analysis to extract the frequency-dependent dielectric function, and a final Fourier transform from frequency to time domain. This time-dependent dielectric function was interpreted as the impulse response of electrons, phonons, or ions based on the Drude–Lorentz theory. The time evolution of electronic oscillators from ice and protein, extracted from low resolution experimental data, were compared. Using higher energy resolution data we have also extracted the time-resolved spectra from excitons in an alkali halide, BaF2. Despite the small scanning transmission electron microscope probe size, delocalization limits the spatial resolution to about 50 nm, which is, nevertheless, better than the millimeter resolution of infrared absorption spectroscopy or Raman spectroscopy.

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.

An innovative approach, combining field-emission scanning electron microscopy (FESEM) with energy dispersive X-ray spectroscopy (EDX) analysis, is presented to investigate the degradation mechanisms affecting tannin-dyed wool. In fact, tannin-dyed textiles are more sensitive to degradation then those dyed with other dyestuffs, even in the same conservation conditions.

FESEM-EDX was first used to study a set of 48 wool specimens (artificially aged) dyed with several raw materials and mordants, and prepared according to historical dyeing recipes. EDX analysis was performed on the surface of wool threads and on their cross-sections. In addition, in order to validate the model formulated by the analysis of reference materials, several samples collected from historical and archaeological textiles were subjected to FESEM-EDX analysis.

FESEM-EDX investigations enabled us to reveal the correlation between elemental composition and morphological changes. In addition, aging processes were clarified by studying changes in the elemental composition of wool from the protective cuticle to the fiber core in cross-sections. Morphological and elemental analysis of wool specimens and of archaeological and historical textiles showed that the presence of tannins increases wool damage, primarily by causing a sulfur decrease and fiber oxidation.

This paper presents a method developed to quantify three-dimensional energy dispersive spectrometry (3D EDS) data with voxel size smaller than the volume from which X-rays are emitted. The influence of the neighboring voxels is corrected by applying recursively a complex quantification, improving thereby the accuracy of the quantification of critically small features. The enhanced quantification method is applied to simulated and measured data. A systematic improvement is obtained compared with classical quantification, proving the concept and the prospect of this method.

In the present work, Pt nanoparticles were produced from a reaction mixture containing a trace amount of cobalt carbonyl salt acting as a shape inducer. Nanoparticle shape evolution during reaction mixture reflux was monitored by characterizing particles extracted from the reaction mixture at different times. It was observed that 5 min of reflux produced spherical nanoparticles, 30 min of reflux produced cube shaped nanoparticles, and 60 min of reflux produced truncated octahedron morphology nanoparticles. It is illustrated that during nanoparticle synthesis the reflux process can provide energy needed for shape transformation from a metastable cube morphology to a truncated octahedron morphology which is thermodynamically the most stable geometry for fcc crystals. An optimization of the reaction reflux is thus needed for isolating metastable shapes.

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.

Cell motility involves a number of strategies that cells use in order to seek nutrients, escape danger, and fulfill morphogenetic roles. Here we present a methodology to quantify morphological changes and their relationship with signaling events from time-lapse imaging microscopy experiments, in order to characterize physiological and pathological processes. To this aim, the stationary spatial pattern of signaling events is determined through an intracellular fluorescent probe, and it is related with the frequency and entity of morphodynamic events, which are in turn quantified through a stochastic approach: two pseudoimages are obtained from a time series of moving cells that describe the probability that a pixel belongs to the cell, and the probability that a pixel is subject to a dynamic event. The simultaneous construction of these maps permits visualization of hot spots of dynamic events, i.e., zones of formation of membrane protrusions and retractions and their relationship with the signaling events reported by the specific probe employed. The method is tested on spontaneous movement of cells, trasfected with redox-sensitive yellow fluorescent protein, in which the distribution of the hot spots and its change upon expression of constitutively active Rac (V12-Rac), is related to the distribution of oxidized spots.

We investigated the structural complexity and texture of the cytoskeleton and nucleus in human mesenchymal stem cells during early phase differentiation into osteoblasts according to the differentiation–induction method: mechanical and/or chemical stimuli. For this, fractal dimension and a number of parameters utilizing the gray-level co-occurrence matrix (GLCM) were calculated based on single-cell images after confirmation of differentiation by immunofluorescence staining. The F-actin and nuclear fractal dimensions were greater in both stimulus groups compared with the control group. The GLCM values for energy and homogeneity were lower in fibers of the F-actin cytoskeleton, indicating a dispersed F-actin arrangement during differentiation. In the nuclei of both stimulus groups, higher values for energy and homogeneity were calculated, indicating that the chromatin arrangement was chaotic during the early phase of differentiation. It was shown and confirmed that combined stimulation with mechanical and chemical factors accelerated differentiation, even in the early phase. Fractal dimension analysis and GLCM methods have the potential to provide a framework for further investigation of stem cell differentiation.

We compared the characteristics of neural cells derived from induced pluripotent stem (iPS) cells from a patient with multiple sclerosis versus neurally differentiated control iPS cells of a healthy individual. The iPS cells were differentiated toward the oligodendrocyte lineage using a four-step protocol established for the differentiation of embryonic stem cells. The resulting cell population was immunostained on day 112 of differentiation for the presence of oligodendrocytes and analyzed by transmission electron microscopy (TEM). Both patient and control samples resembled a mixed population of neural cells rather than oligodendroglia of high purity, including neural stem cell-like cells and possibly oligodendrocytes demonstrable by TEM.

Micro-morphometry has substantially gained ground in the field of phytolith analysis, but the comparability of results is limited due to the use of different methods. This paper presents a new, user-friendly method based on open-source software (FIJI) that is proposed as a step towards the introduction of a standard method. After obtaining a mask of a phytolith by making a digital drawing, 27 commonly used variables of size and shape are measured automatically. This method is not only useful for phytolith analysis, but may also be used for other fields of morphometric research. Users can furthermore customize the software tool when additional variables are required.

A test material for routine performance evaluation of energy-dispersive X-ray spectrometers (EDS) is presented. It consists of a synthetic, thick coating of C, Al, Mn, Cu, and Zr, in an elemental composition that provides interference-free characteristic X-ray lines of similar intensities at 10 kV scanning electron microscope voltage. The EDS energy resolution at the C-K, Mn-Lα, Cu-Lα, Al-K, Zr-Lα, and Mn-Kα lines, the calibration state of the energy scale, and the Mn-Lα/Mn-Kα intensity ratio as a measure for the low-energy detection efficiency are calculated by a dedicated software package from the 10 kV spectrum. Measurements at various input count rates and processor shaping times enable an estimation of the operation conditions for which the X-ray spectrum is not yet corrupted by pile-up events. Representative examples of EDS systems characterized with the test material and the related software are presented and discussed.

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.