Transmission electron microscopy (TEM) images of beam sensitive weak-phase objects such as biological cryo samples usually show a very low signal-to-noise ratio. These samples have almost no amplitude contrast and instead structural information is mainly encoded in the phase contrast. To increase the sample contrast in the image, especially for low spatial frequencies, the use of phase plates for close to focus phase contrast enhancement in TEM has long been discussed. Electrostatic phase plates are favorable in particular, as their tunable potential will allow an optimal phase shift adjustment and higher resolution than film phase plates as they avoid additional scattering events in matter. Here we show the first realization of close to focus phase contrast images of actin filament cryo samples acquired using an electrostatic Zach phase plate. Both positive and negative phase contrast is shown, which is obtained by applying appropriate potentials to the phase plate. The dependence of phase contrast improvement on sample orientation with respect to the phase plate is demonstrated and single-sideband artifacts are discussed. Additionally, possibilities to reduce contamination and charging effects of the phase plate are shown.

One of the few conclusions known about chromosome structure is that Mg2+ is required for the organization of chromosomes. Scanning electron microscopy is a powerful tool for studying chromosome morphology, but being nonconductive, chromosomes require metal/carbon coating that may conceal information about the detailed surface structure of the sample. Helium ion microscopy (HIM), which has recently been developed, does not require sample coating due to its charge compensation system. Here we investigated the structure of isolated human chromosomes under different Mg2+ concentrations by HIM. High-contrast and resolution images from uncoated samples obtained by HIM enabled investigation on the effects of Mg2+ on chromosome structure. Chromatin fiber information was obtained more clearly with uncoated than coated chromosomes. Our results suggest that both overall features and detailed structure of chromatin are significantly affected by different Mg2+ concentrations. Chromosomes were more condensed and a globular structure of chromatin with 30 nm diameter was visualized with 5 mM Mg2+ treatment, while 0 mM Mg2+ resulted in a less compact and more fibrous structure 11 nm in diameter. We conclude that HIM is a powerful tool for investigating chromosomes and other biological samples without requiring metal/carbon coating.

The size of gold nanoparticles (AuNPs) can influence various aspects of their cellular uptake. Light microscopy is not capable of resolving most AuNPs, while electron microscopy (EM) is not practically capable of acquiring the necessary statistical data from many cells and the results may suffer from various artifacts. Here, we demonstrate the use of a fast EM method for obtaining high-resolution data from a much larger population of cells than is usually feasible with conventional EM. A549 (human lung carcinoma) cells were subjected to uptake protocols with 10, 15, or 30 nm diameter AuNPs with adsorbed serum proteins. After 20 min, 24 h, or 45 h, the cells were fixed and imaged in whole in a thin layer of liquid water with environmental scanning electron microscopy equipped with a scanning transmission electron microscopy detector. The fast preparation and imaging of 145 whole cells in liquid allowed collection of nanoscale data within an exceptionally small amount of time of ~80 h. Analysis of 1,041 AuNP-filled vesicles showed that the long-term AuNP storing lysosomes increased their average size by 80 nm when AuNPs with 30 nm diameter were uptaken, compared to lysosomes of cells incubated with AuNPs of 10 and 15 nm diameter.

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.

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.

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.

Strigomonas culicis (previously referred to as Blastocrithidia culicis) is a monoxenic trypanosomatid harboring a symbiotic bacterium, which maintains an obligatory relationship with the host protozoan. Investigations of the cell cycle in symbiont harboring trypanosomatids suggest that the bacterium divides in coordination with other host cell structures, particularly the nucleus. In this study we used light and electron microscopy followed by three-dimensional reconstruction to characterize the symbiont division during the cell cycle of S. culicis. We observed that during this process, the symbiotic bacterium presents different forms and is found at different positions in relationship to the host cell structures. At the G1/S phase of the protozoan cell cycle, the endosymbiont exhibits a constricted form that appears to elongate, resulting in the bacterium division, which occurs before kinetoplast and nucleus segregation. During cytokinesis, the symbionts are positioned close to each nucleus to ensure that each daughter cell will inherit a single copy of the bacterium. These observations indicated that the association of the bacterium with the protozoan nucleus coordinates the cell cycle in both organisms.

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.

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.

Elemental analysis of dental hard tissues is of importance. The aim of this study is to evaluate X-ray microanalysis (XRMA) of bovine enamel in a scanning electron microscope (SEM) with different coatings. The buccal surface of bovine incisors was polished flat, one-third was coated with carbon, one-third with gold, leaving one-third uncoated for XRMA in an SEM equipped with an energy-dispersive microanalysis system. The elements oxygen, sodium, magnesium, phosphorous, chlorine, potassium, and calcium were analyzed using their respective characteristic K X-ray series. Comparisons were made with analyses of glass produced by fusion of the bovine enamel, showing that oxygen analyses using the K X-ray series are reliable and preferable to calculating oxygen by stoichiometry for natural enamel. For the gold-coated and uncoated analyses, carbon was also measured using the K X-ray series. Small area Analyses in small areas (80 × 80 μm) in variable pressure-SEM mode with low vacuum (20 Pa), without any coating, midway between 40 μm wide gold lines 140 μm apart to avoid build-up of electrostatic charge is the preferred method, especially if carbon is included in the analysis. The analyses of bovine enamel are sufficiently reproducible to be regarded as quantitative for all elements except carbon.

Sample preparation and imaging techniques for scanning electron microscopy (SEM) of dehydrated dental samples can hinder the structural analyses. This study qualitatively evaluated images obtained with two different protocols of SEM preparation and analysis to assess the dentine adhesive interface. The crown and root dentine of 12 bovine incisors were subjected to cementation with the resin cement RelyX U100 or RelyX ARC/SBMP (n = 6). After storage for 7 days in a moist environment at 37 ± 1°C, the dentine samples were dehydrated in an ascending alcohol series, and three specimens from each group were coated with gold or carbon and examined in a high-vacuum (JEOL JSM—6360LV, 10 kV) or low-vacuum (FEI Quanta 200F, 15–30 kV) microscope. Images were obtained at magnifications between 50 and 2,000×, but with different working distances. The use of high vacuum for carbon and gold coating and SEM visualization led to cracks in the samples. A small number of cracks can be described in the specimens subjected to the low-vacuum technique. The protocol for SEM imaging in low vacuum was considered more appropriate for preservation of the integrity of the evaluated structures.

Species of Haliotis (abalone) show high variety in structure and mineralogy of the shell. One of the European species (Haliotis tuberculata) in particular has an unusual shell structure in which calcite and aragonite coexist at a microscale with small patches of aragonite embedded in larger calcitic zones. A detailed examination of the boundary between calcite and aragonite using analytical microscopies shows that the organic contents of calcite and aragonite differ. Moreover, changes in the chemical composition of the two minerals seem to be gradual and define a micrometric zone of transition between the two main layers. A similar transition zone has been observed between the layers in more classical and regularly structured mollusk shells. The imbrication of microscopic patches of aragonite within a calcitic zone suggests the occurrence of very fast physiological changes in these taxa.

Bumblebees need to keep bodily homeostasis and for that have an efficient system of excretion formed by the Malpighian tubules, ileum, and rectum. We analyzed the excretory organs of Bombus morio, a bee without rectal pads. In addition, we analyzed the rectal epithelium of Melipona quadrifasciata anthidioides which has rectal pads. The Malpighian tubules exhibited two cell types and the ileum four types. However, comparative analysis of the rectum showed that only cells of the anterior region of the rectal epithelium of B. morio are structurally distinct. We suggest that cells of the Malpighian tubules of B. morio have an excretory feature and that cells of ileum have different functions, such as ion absorption and water, organic compound, and protein secretion. In addition, only the anterior region of the rectum of B. morio showed characteristic absorption. We suggest that Malpighian tubules participate in the excretion of solutes and that the ileum and rectal epithelium are responsible for homeostasis of water and solutes, compensating for the absence of rectal papillae. These results contribute to our understanding of the morphophysiology of the excretory organs of bees without rectal pads.

In this work the leaf anatomy of three species of Ficus section Americanae (Miq.) Miq. from Brazil, whose leaves and latex are used in folk medicine is reported. The work was carried out using light and scanning electron microscopy in order to characterize these species and to evaluate their taxonomic significance, and also contribute to the quality control of their ethnodrugs. The three species (Ficus cyclophylla, Ficus elliotiana, and Ficus caatingae) showed hypostomatic leaves, anomocytic stomata, straight epidermal cell outlines, and a dorsiventral mesophyll. Some micro-morphological characters such as density and distribution of epicuticular waxes, glandular trichomes, the length and width of stomata, as well as the palisade of mesophyll and petiole outlines proved to be the most useful and distinctive characters for the separation of species. These may contribute as additional support for the taxonomy of the section and for the quality control of their ethnodrugs.

A multiscale approach combining phase-contrast X-ray micro- and nanotomography is applied for imaging a Cretaceous fossil inflorescence in the resolution range from 0.75 μm to 50 nm. The wide range of scale views provides three-dimensional reconstructions from the external gross morphology of the inflorescence fragment to the finest exine sculptures of in situ pollen. This approach enables most of the characteristics usually observed under light microscopy, or with low magnification under scanning and transmission electron microscopy, to be obtained nondestructively. In contrast to previous tomography studies of fossil and extant flowers that used resolutions down to the micron range, we used voxels with a 50 nm side in local tomography scans. This high level of resolution enables systematic affinities of fossil flowers to be established without breaking or slicing specimens.