Visualization of the native shape of bodipy‐labeled DNA in Escherichia coli by correlative microscopy

The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL‐14‐dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high‐resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ～ 10 mmol/kg in area of 0.1 μm², which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase‐plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase‐plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.     

Ultrastructural and electron spectroscopic analyses of cyanobacteria and bacteria

The extracellular sheath material and some intracellular cell components of cyanobacteria and phosphate-accumulating sewage bacteria were analysed by electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). The specimens were embedded in water-soluble Nanoplast resin without any previous fixation and ultrathin sections were examined in a Zeiss CEM 902 microscope. A high sulphur content was detected in the inner sheath of the cyanobacterium Gloeothece. The elemental composition of some cell components and inclusion bodies, such as carboxysomes and cyanophycin, was determined by ESI and EELS. In addition, the phosphate content in specific granules of phosphate-accumulating sewage bacteria was estimated by EELS and nuclear magnetic resonance spectroscopy.     

Ultrastructural localization of titanium in chemically modified chrysotile asbestos

The ultrastructural localization of Ti in chemically modified chrysotile fibres has been studied. TiCl₃-treated fibres were analysed by electron energy-loss spectroscopy and electron spectroscopic imaging (ESI) along directions parallel and perpendicular to the fibre axis. ESI of ultrathin sections of conventionally embedded chrysotile fibres was made possible with the use of a cryo-stage. The ESI images observed in fibre cross-sections revealed that the Ti is located not only at the surface of the fibres but also inside the fibril tubes. This combination of microanalysis and imaging in the analytical electron microscope is the most suitable technique for relating the microchemistry and microstructure on a submicrometre scale.     

Light and transmission electron microscopic structure of skin glands and dermal scales of a caecilian amphibian Gegeneophis ramaswamii,with a note on antimicrobial property of skin gland secretion

Amphibian skin secretions contain a variety of bioactive compounds that are involved in diverse roles such as communication, homeostasis, defence against predators, pathogens, and so on. Especially, the caecilian amphibians possess numerous cutaneous glands that produce the secretory material, which facilitate survival in their harsh subterranean environment. Inspite of the fact that India has a fairly abundant distribution of caecilian amphibians, there has hardly been any study on their skin and its secretion. Herein, we describe, using light microscopy and electron microscopy, two types of dermal glands, mucous and granular, in Gegeneophis ramaswamii. The mucous glands are filled with mucous materials. The mucous‐producing cells are located near the periphery. The granular glands are surrounded by myoepithelial cells. A large number of granules of different sizes are present in the lumen of the granular gland. The granule‐producing cells are present near the myoepithelial lining of the gland. There are small flat disk‐like dermal scales in pockets in the transverse ridges of the posterior region of the body. Each pocket contains 1–4 scales of various sizes. Scanning electron microscopic (SEM) study of the skin surface showed numerous funnel‐shaped glandular openings. The antibacterial activity of the skin secretions was revealed in the test against Escherichia coli, Klebsiella pneumoniae, and Aeromonas hydrophila, all gram‐negative bacteria. SEM analyses confirm the membrane damage in bacterial cells on exposure to skin secretions of G. ramaswamii.     

Confocal laser scanning microscopic analysis of ectopic sublingual gland‐like tissue inside the hamster submandibular gland

Based on its histochemical properties, the secretory portion of the hamster submandibular gland has been classified as seromucous cells. The presence of endogenous peroxidase (PO) reaction was shown in the nuclear envelope, cisternae of endoplasmic reticulum and Golgi apparatus. The 3,3′‐diaminobenzidene, tetrahydrochloride (DAB) method revealed bipartite secretory granules containing a PO‐positive dense core surrounded by a less dense halo in these cells. In the present investigation, serous and mucous‐like cells were found in resin‐embedded semi‐thin sections of the DAB‐reacted hamster submandibular gland. These sections were already on glass slides for routine light microscopic observations, therefore electron microscopic analysis could be unrealizable. We then used reflectance‐mode confocal laser scanning microscopy to visualize additional sites of PO activity as detected in these sections. Using this approach, we found mucous cells with PO activity‐negative secretory granules and seromucous cells with PO activity‐positive spot‐like secretory granules of the regular sublingual gland most frequently adjacent to the serous cells with typical electron‐dense secretory granules. These cells clearly differ from the seromucous cells with bipartite secretory granules and the granular duct cells with typical electron‐dense secretory granules of the hamster submandibular gland. Additionally, secretory endpieces of the ectopic sublingual gland‐like tissue empty into the duct of the hamster submandibular gland lobule. Thus, our findings suggest that a mass of sublingual gland tissue extends into the hamster submandibular gland during its development, and PO may be synthesized and secreted into the same duct. Microsc. Res. Tech. 76:1284–1291, 2013. © 2013 Wiley Periodicals, Inc.     

Nonmarski differential-interference contrast microscopy in transillumination: its use on unstained or stained sections, smears, and wet mounts, or on fluorochromed sections and cell-culture monolayers

Unstained, lightly stained and conventionally stained microtome tissue sections of two different thicknesses (ca. 4 μm and 1 μm) and also unstained or stained wet mounts of cells were photographed under the microscope using bright-field, positive phase contrast, and Nomarski differential-interference contrast (DIC) in transillumination. The photomicrographs were critically compared. It was found that the density of various stains did not adversely affect the better resolution of the DIC image (as compared to the bright-field image); however, Optical sectioning' of darkly stained objects is not possible. Unstained or stained smears of blood or of epithelial cells of buccal mucosa were examined with DIC in transillumination, then after certain preparatory techniques, the same preparation was examined in the scanning electron microscope, and finally the same areas of the slide were viewed with DIC in epiillumination. Particular attention was given to structures (nuclei and cytoplasm) which appeared in positive or negative relief in the photomicrographs taken by the various techniques. It was concluded that the optically more dense nucleus which always appeared in positive relief by the various methods of examination, was in fact geometrically raised from the surrounding cytoplasm. Acridine orange (AO) stained cell-culture monolayers and H and E stained sections were examined under a fluorescence microscope with DIC optics. By comparing photographs which had been taken with DIC, epi-fluorescence or fluorescence in transillumination, and DIC-fluorescence, it was concluded that the DIC image, which had been superimposed on the fluorescence image, contributed a definite gain in information. Some common errors in the interpretation of the DIC image are discussed; methods of avoiding improper use of equipment are given. The conclusion is drawn that the DIC system is superior to positive and negative phase contrast for the examination of a variety of unstained or stained preparations. Therefore, this method can be used to advantage not only for the examination of unstained preparations, but also on some specimens which have been routinely stained or fluorochromed.     

Defense reactions of <i>Dermatobia hominis</i> (Diptera: Cuterebridae) larval hemocytes

The defense reactions against biological (Histoplasma capsulatum and Escherichia coli) and non-biological materials (China ink and nylon thread) were tested in vivo in third instar larvae of Dermatobia hominis. The cellular defense performed by larval hemocytes was observed under electron microscopy. China ink particles were phagocytosed by granular cells 5 h after injection. E. coli cells were internalized by granular cells as early as 5 min after injection and totally cleared 180 min post-injection, when many hemocytes appeared disintegrated and others in process of recovering. H. capsulatum yeasts provoked, 24 h after being injected, the beginning of nodule formation. Nylon thread was encapsulated 24 h after the introduction into the hemocoel. Our results suggest that granular cells were the phagocytic cells and also the responsible for the triggering of nodule and capsule formation. In the presence of yeasts cells and nylon thread, they released their granules that chemotactically attracted the plasmatocytes that on their turn, flattened to surround and isolate the foreign material.     

Limitations of beam damage in electron spectroscopic tomography of embedded cells

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two‐dimensional (2D) core‐edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic‐embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic‐embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10⁶ e^?/nm², no significant loss of phosphorus or nitrogen was observed for doses as high as 10⁸ e^?/nm². The oxygen content decreased from 25 ± 2 to 9 ± 2 atomic percent at an electron dose of 10⁴ e^?/nm², which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10⁸ e^?/nm², and a lateral shrinkage of 9.5 ± 2.0% occurred from 2 × 10⁴ to 10⁸ e^?/nm². At doses above 10⁷ e^?/nm², damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high‐pressure frozen and freeze‐substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.     

Contribution of energy-filtering TEM to the detection of calcium: Application to mast cells

The ultrastructural distribution and quantification of calcium in mast cells prepared by anhydrous processing was investigated by energy-filtering transmission electron microscopy (EFTEM) using a Zeiss 902 electron microscope. Optimal conditions for calcium detection were determined using inorganic (calcium phosphate) and organic (calcium-loaded chelex beads) standards with known amounts of calcium. Electron energy-loss spectroscopy (EELS) revealed calcium at the L_2,3 edge and also at the M_2,3 edge for all specimens examined. Comparison with X-ray microanalysis confirmed the results obtained with EELS. Electron spectroscopic imaging (ESI) was applied for mapping calcium both in standards and in cells and we showed that mast cell granules were the main site of calcium localization. Although, results have shown that a combination of analytical techniques is required to obtain reliable results.     

Investigation and use of plasmon losses in energy-filtering transmission electron microscopy

The electron spectroscopic imaging (ESI), diffraction (ESD) and different types of electron energy-loss spectroscopy (EELS) modes in an energy-filtering transmission electron microscope can all be used for the investigation and analytical use of plasmon losses. Shifts of plasmon losses caused by differences in composition can be detected with an accuracy of 0.1 eV by parallel-recorded EELS (PEELS). The dispersion of plasmon losses and the cut-off angle θ_c can be observed by angle-dispersive EELS and by recording spectra at different scattering angles θ. ESD patterns with a selected energy window width of 1 eV enable the dispersion and its anisotropy to be imaged by characteristic intensity distributions between the primary beam and the first Bragg diffracted beams. The ESI mode can be used for the selective imaging of precipitates and for the investigation of the excitation volume of plasmons in small particles.     

Tracing DiO-labelled tumour cells in liver sections by confocal laser scanning microscopy

Investigating rare cellular events is facilitated by studying thick sections with confocal laser scanning microscopy (CLSM). Localization of cells in tissue sections can be done by immunolabelling or by fluorescent labelling of cells prior to intravenous administration. Immunolabelling is technically complicated because of the preservation of antigens during fixation and the problems associated with the penetration of the antibodies. In this study, an alternative and simple approach for the labelling of cells in vitro with the fluorescent probe DiO and its subsequent application in vivo will be outlined. The method was applied to trace DiO‐labelled colon carcinoma cells (CC531s) in 100 µm thick liver sections. In vitro and in vivo experiments revealed that DiO‐labelling of CC531s cells had no cytotoxic or antiproliferative effect and the cells preserved their susceptibility towards hepatic NK cells or Kupffer cells. In addition, DiO remained stable for at least 72 h in the living cell. DiO‐labelled CC531s cells could be traced all over the tissue depth and anti‐metastatic events such as phagocytosis of tumour cells by Kupffer cells could be easily observed. In situ staining with propidium iodide (nucleic acids) or rhodamine‐phalloidin (filamentous actin) resulted in additional tissue information. The data presented improved the understanding of the possible effects of the vital fluorescent probe DiO on cell function and provided a limit of confidence for CLSM imaging of DiO‐labelled cells in tissue sections.     

Low-temperature SEM for detection of fungicide activity

Low-temperature scanning electron microscopy (LTSEM) combined with cryopreparation methods provided images of well-preserved biological surfaces and structures on a routine basis. Fractures of wheat leaves revealed epidermal and parenchymatous cells and masses of fungal hyphae growing in intercellular spaces. Freeze-fractured plant cells contained haustoria of the brown rust fungus Puccinia triticina. Extrahaustorial matrices were clearly distinguishable and at infection sites granular material was found. Activity of the triazole fungicide cyproconazole was mainly directed towards fungal hyphae and sporogenic tissue, resulting in a stronger branching and swelling of hyphal tips and collapse of fungal cells. Cryofixation methods combined with the use of a cryopreparation unit (Balzers SCU 020) were more reliable in interpreting the observed biological events through easier discrimination between evidence and artefacts. More advantages in the use of the cryopreparation unit are described in detail.     

General morphology and ultrastructure of the female reproductive apparatus of Trichomalopsis shirakii crawford (Hymenoptera,Pteromalidae)

The morphology and ultrastructure of the female reproductive system were examined for a larval–pupal parasitoid Trichomalopsis shirakii Crawford of Oulema oryzae Kuwayama using light and electron microscopes. The reproductive system includes two ovaries, two pairs of accessory glands, an unbranched venom gland, a large venom reservoir and a Dufour gland. Each ovariole contains follicles and oocytes at different stages of maturation. A fibrous layer covers the surface of mature egg. The accessory glands are made up of a layer of secretory cells surrounded by muscle fibers. In these secretory cells, numerous mitochondria, electron‐dense secretory granules and vesicles filled with dense granular particles are present. These granular particles appear as virus‐like particles (VLPs). The venom gland consists of a single layer of secretory cells which are organelle rich with abundant rough endoplasmic reticulum, mitochondria and vesicular organelles, a layer of duct cells and an inner intima. The reservoir consists of a muscular sheath, epidermal cells with few organelles and an intima layer. The Dufour gland has a relatively large lumen surrounded by a single layer of columnar epithelial cells which are characterized by clusters of smooth endoplasmic reticulum and lipid droplets. Aside from the venom, the fibrous layer coating the egg and the granular particles which may be VLPs have been discovered in our study. They may serve as one of the parasitoid‐associated factors in their host–parasitoid relationship and play a role in host immune suppression. Microsc. Res. Tech. 79:625–636, 2016. © 2016 Wiley Periodicals, Inc.     

Conventional transmission electron microscopy (CTEM) of unstained,melamine-embedded frog photoreceptor membranes

Melamine-formaldehyde, a universally used technical aminoplast, was recently introduced as a water-soluble embedding-medium for electron microscopy (Bachhuber & Frösch, 1983). When processed with this new technique, the membranes of aldehyde-fixed, unstained frog photoreceptor outer segments show remarkable contrast and detail in conventional TEM bright field imaging. Through-focus-series, showing true contrast reversal, indicate that images from defocused, unstained melamine sections are generated primarily by phase contrast.     

Probing nuclear ultrastructure by electron spectroscopic imaging

Mammalian nuclei are complex organelles containing many functionally distinct nucleoprotein and protein particles in the size range 20–30 nm. This complexity hinders the study of structure-function relationships within the mammalian nucleus. Element-specific mapping using the energy-filtered transmission electron microscope can provide novel information on protein and nucleic acid density within structures, facilitating the identification of biochemical heterogeneity within morphologically similar structures. We demonstrate that imaging phosphorus, nitrogen and carbon can be useful in the characterization of protein and nucleoprotein structures within the nucleus. Additionally, electron spectroscopic imaging (ESI) may be used to map the distribution of stains relative to unstained material when biochemical-specific staining protocols, such as EDTA-regressive staining of RNA with uranyl acetate, are used. Relative mass may also be determined from ESI images and can be combined with elemental information further to distinguish biological constituents. Using this approach, heterochromatin was found to be variable in nucleic acid content although the morphology appeared relatively homogeneous. ESI shows substantial promise for the investigation of structure–function relationships in biological specimens.     

The application of energy-filtering electron microscopy for the cytochemical localization of Ca2+ -ATPase activity in synaptic terminals

The energy-filtering electron microscopical modes of electron energy-loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) have been applied to the cytochemical detection of Ca²⁺ -ATPase activity in synaptic terminals in the brain of a cichlid fish. Using a recently developed modification of an enzyme-histochemical method, cerium phosphate was precipitated as a marker of high-affinity Ca²⁺ -ATPase activity. This is considered to be a marker for the plasmalemma-bound calcium pump, an enzyme which plays a crucial role in the regulation of the cytoplasmic calcium concentrations and therefore of the reactivity of nerve cells. High-affinity Ca²⁺ -ATPase activity is located preferentially at the inner side of synaptic plasma membranes and enables a discrimination of different types of synapse. It is only by using EELS and ESI that the very small amounts of high-affinity Ca²⁺ -ATPase reaction product can be analysed reliably and located precisely. These new electron microscopical techniques offer powerful tools for cytochemical studies.     

Expression of caveolin-1 in rat Leydig cells

Caveolin-1, the first member of caveolin family reported, is recognized as the structural component of caveola, a plasma membrane invagination or vesicles that are a subcompartment distinct from clathrin-coated pits. This protein is also known to be involved in cholesterol trafficking.
The aim of this study was to determine the expression of caveolin-1 in adult rat Leydig cells. Testis sections incubated with an antibody to caveolin-1 showed, by immunohistochemistry, a moderate number of Leydig cells with different degrees of immunoreaction and a strong reaction in endothelial cells and in the lamina propia of seminiferous tubules. Caveolin-1 was detected in the cell cytoplasm with a granular pattern and on the cell surface of Leydig cells cultured 24 h on uncoated, laminin-1 or type IV collagen coated coverslips. We also observed a milder reaction in 3 h cultures. Immunoreaction was also detected in Leydig cells with an antibody to tyrosine-phosphorylated caveolin-1. By double immunofluorescent technique, we observed co-localization of caveolin-1 and 3β-hydroxysteroid dehydrogenase. Western blot analysis revealed a band of about 22 kDa molecular weight that was recognized with both caveolin-1 and tyrosinephosphocaveolin-1 antibodies. Caveolin-1 is one of a few proteins with a demonstrated ability to bind cholesterol in vivo. In this context, the presence of caveolin-1 in Leydig cells may be related to cholesterol traffic -a rate limiting step in steroid biosynthesis.     

Ultrastructural comparison of ion beam and radiofrequency plasma etching effects on biological tissue sections

Three dry etching techniques (Ar+ ion beam, O₂+ ion beam, O₂ radiofrequency electrodeless discharge) were compared with respect to preferential etching and damage to the ultrastructure of glutaraldehyde-fixed Epon-embedded frog skeletal muscle sections. SEM and TEM studies were performed on both unstained and stained (osmium tetroxide, uranyl acetate) sections. Etching effects were observed to differ for the various ion beam or plasma etching techniques. Whereas selective retention of electron dense structures (e.g. Z lines, nuclear heterochromatin) was observed for oxygen plasma etching, preferential etching of these components was observed using O₂+ ion beam bombardment. Selectively etched Z lines and etch-resistant nucleoli were observed for both reactive (O₂+) and inert (Ar+) ion beam sputtering after sufficiently high ion doses. The above suggest that selective etching under keV ion beam irradiation is related more to physical sputtering processes (momentum transfer) than to the chemical reactivity of the incident ion. Heavy metal post-fixation and staining had no qualitative effect on the nature of the selective etching phenomena. The above findings are significant in that they potentially influence both electron and ion microprobe measurements of etched biological specimens.     

Energy-filtered transmission electron microscopy of biological samples on highly transparent carbon nanomembranes

Ultrathin carbon nanomembranes (CNM) comprising crosslinked biphenyl precursors have been tested as support films for energy-filtered transmission electron microscopy (EFTEM) of biological specimens. Due to their high transparency CNM are ideal substrates for electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) of stained and unstained biological samples. Virtually background-free elemental maps of tobacco mosaic virus (TMV) and ferritin have been obtained from samples supported by ∼1 nm thin CNM. Furthermore, we have tested conductive carbon nanomembranes (cCNM) comprising nanocrystalline graphene, obtained by thermal treatment of CNM, as supports for cryoEM of ice-embedded biological samples. We imaged ice-embedded TMV on cCNM and compared the results with images of ice-embedded TMV on conventional carbon film (CC), thus analyzing the gain in contrast for TMV on cCNM in a quantitative manner. In addition we have developed a method for the preparation of vitrified specimens, suspended over the holes of a conventional holey carbon film, while backed by ultrathin cCNM.     

Structural observations and biomechanical measurements of clarinet reeds made from Arundo donax

Plant anatomy was examined for two clarinet reeds made out of Arundo donax by different means of microscopy: light microscopy, low‐energy secondary electron scanning electron microscopy (SEM), backscattered electron SEM, and helium ion microscopy (HiM). The local indentation hardness H_IT and Young's modulus E_IT of different tissues on their cross sections were measured. A vascular bundle (Vb) (H_IT = 60–100 MPa, E_IT = 1,500–2,000 MPa) that includes soft tissues of phloem and xylem and a vascular bundle sheath (Bs) (H_IT = 300–500 MPa, E_IT = ～7,000 MPa) form a pipe of the strong string along the longitudinal direction of the cane. This Vb/Bs string is connected transversally with a net of thin cell‐walls of parenchyma cells (Pa) (H_IT = 70–200 MPa, E_IT = 2,000–3,000 MPa) that also range along the longitudinal direction of the cane. It was turned out that the acoustic quality of a reed is mainly ascribed to the shape and configuration of Vb and the size of Pa. A reed where Vb bundles with continuous Bs rings are homogeneously distributed with higher proportion among a softer network of small Pa cells enables musical performance.