Morphological and structural studies of early mineral formation in enamel of rat incisors by electron spectroscopic imaging (ESI) and electron spectroscopic diffraction (ESD)

Morphological and structural analysis of the earliest stage of crystal formation in enamel of rat incisors, by use of energy filtering transmission electron microscopy (EFTEM), has shown needlelike crystallites with a dotlike substructure. We conclude that these dots (nanometer-sized particles) have developed at nucleating, active sites along the non-collagenous matrix proteins in enamel. Calcium and phosphate groups are bound at such active sites and develop to nuclei, which grow to these stable dots (nanometer-sized particles). The dots coalesce rapidly in longitudinal direction, along the matrix proteins, with neighbouring dots to form parallel arranged needlelike crystallites. These needles grow and coalesce in lateral directions to ribbon-platelike crystallites. In enamel most of the organic substance becomes decomposed and transported to the ameloblasts. Consequently, the ribbon-platelike crystallites can coalesce to form much thicker (hydroxy)-apatite crystals than in dentine. Already in the earliest stage of crystal formation the mineral chains of dots (nanometer-sized particles) and the needlelike crystallites show a parallel orientation in the direction of the c-axis of hydroxyapatite. This is supported by the texture of the 002 reflections in the corresponding electron spectroscopic diffraction patterns (ESD), which appear as the first Bragg reflections.     

Investigation of the early mineralisation on collagen in dentine of rat incisors by quantitative electron spectroscopic diffraction (ESD)

The earliest crystallites in dentine appear as chains of dots in ultra-thin sections viewed by transmission electron microscopy. These dots rapidly coalesce along the longitudinal directions of the collagen microfibrils to form needle-like structures that coalesce preferentially in lateral directions to form ribbon-like or plate-like crystallites. This morphological interpretation is supported by line-scans of the corresponding zero-loss filtered electron spectroscopic diffraction patterns, which demonstrate the crystalline structure of the dentine mineral (apatite). The intensity ratio of the Debye-Scherrer rings of the characteristic Bragg-reflections (002 to 300, together with 1 or 2 unresolved reflections) shows a maximum in the region of early chain-like and needle-like crystallites, decreasing with maturation of the dentine mineral to the ribbon-plate-like crystallites. Detailed investigations using line-scans of the zero-loss filtered electron spectroscopic diffraction patterns through the dentine zone show that the intensity ratio found near the mineralisation front is repeated 3–5 times at distances of about 10–20 m. This may represent a circadian pattern of mineralisation corresponding to light microscopically visible incremental lines in dentine.     

Localization of calcium in the cyanobiont and gonidial zone ofCycas revoluta Thunb. by microelectrodes,chlorotetracycline, electron spectroscopic imaging and electron energy loss spectroscopy

Summary Ionic calcium concentration was measured in the gonidial zone of fresh coralloid roots by means of calcium microelectrodes. It was 10⁻⁶ M in the apical segments of coralloid roots and increased to 10⁻⁵ M in the gonidial zones of median and basal segments. Loosely membrane-bound calcium was evidenced by using chlorotetracycline (CTC) or ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and CTC, in cell walls of columnar cells ofCycas and in the cytoplasm of cyanobiont. Sub-cellular localization of calcium was obtained by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) analyses applied at transmission electron microscopy on thin, unstained sections of gonidial zone of coralloid roots. By means of these techniques, bound-calcium was detected inside the mucilage of apical and median segments whereas, in the basal segments, it was completely absent. In the heterocysts of apical segments of coralloid, calcium was localized on the envelope, cell walls, thylakoids and cyanophycin granules. In the gonidial zone of the basal segments, dead or degenerating heterocysts completely lacked calcium. Therefore, the high ionic calcium amounts detected in the gonidial zone of median and basal segments could represent a minor calcium uptake by the cells or release by lysed ones. The decreases in nitrogenase activity recorded in the median and basal segments of the coralloid roots paralleled the decrease in calcium amount in heterocyst envelope.     

High resolution microanalysis for phosphorus in Golgi complex beads of insect fat body tissue by electron spectroscopic imaging

Golgi complex beads are 10 nm particles arranged in rings on the smooth forming face of the Golgi complex that stain specifically with bismuth in arthropod cells. In vitro experiments with biological molecules spotted on to cellulose acetate strips indicated that bismuth bound to the beads through phosphate groups. We could detect a weak phosphorus signal from the beads using a new technique called electron spectroscopic imaging that is capable of very high spatial resolution (0.3–0.5 nm) and sensitivity (50 atoms of phosphorus). Detection was not obscured by tissue staining with bismuth or uranyl acetate or by using an inorganic buffer (Na cacodylate). Localization of phosphorus was greatly improved by using colour-enhanced computer pictures of the electron spectroscopic images and quantitating the images. The results indicate that the phosphorus content of the beads is large enough to account for their bismuth reactivity.     

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 µm, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.     

Ultrastructural variations in Microcystis aeruginosa (Chroococcales,Cyanophyta) during a surface bloom induced by high incident light irradiance

ABSTRACT

We evaluated the ultrastructural variations occurring during a surface bloom in Microcystis aeruginosa Kützing PCC 7005, by comparing cultures exposed to high incident light irradiance (90 µmol photons m^-2 s^-1) and to low irradiance (9 µmol photons m^-2 s^-1). At 12h, and 15 and 30 days in culture, the cells were fixed in glutaraldehyde and subjected to cytochemical assays. Exposure to high irradiance induced structural changes in the cell wall, and differences in the photosynthetic apparatus and granule reserves. After 15 days of high irradiance, the following features were observed: a reduced number of thylakoids and changes in their arrangement; high accumulation of glycogen, poly-beta-hydroxybutyrate, lipid bodies and cyanophycin; conspicuous structural changes in the cell wall. At day 30 of high irradiance, the viable cells had few thylakoids, arranged parallel to each other and perpendicular to the plasma membrane, and abundant glycogen. Nitrogen, calcium and phosphorus, as detected by electron spectroscopic imaging, were present in different sites of cells grown at low and high irradiance. The results of this study, together with previous data on variation in Fe-superoxide dismutase (Canini et al., 2001a), suggest that defense mechanisms against high and prolonged irradiance only operate for a short time.     

The dynamics of exoskeletal-epidermal structure during molt in juvenile lobster by electron microscopy and electron spectroscopic imaging

The exoskeletal-epidermal complex of juvenile lobsters at various stages throughout the molt cycle was examined by conventional electron microscopy, freeze-etch replicas, and electron spectroscopic imaging. This latter technique which enables the direct localization of atomic elements superimposed over morphological fine structure has been applied to this tissue complex to determine the spatial distributions and interrelationships of calcium, phosphorus, and sulphur. Chitin microfibril assembly is visualized in thin sections as occurring at the surface of apical membrane plaques which in freeze-etch replicas invariably possess a rich distribution of intramembrane particles on both P and E faces. In early stages of mineralization the exo- and endocuticular zones of the exoskeleton possess a dense Ca-containing lamellar repeat. These bands are unrelated to the helicoidal arrangement of chitin microfibrils. At later stages of development mineral deposits occur within the exocuticle and advance through to the endocuticle. These deposits align with chitin microfibrils and exhibit a helicoidal pattern. Morphological and chemical alterations associated with mineralization and demineralization of the exoskeleton are discussed.     

Electron-energy-loss spectroscopic imaging of calcium and nitrogen in the cell walls of apple fruits

Changes in texture are an integral part of ripening in most fleshy fruits and these changes are thought to be determined, primarily, by alterations in cell wall structure. Electron energy loss spectroscopy (EELS) imaging was used to obtain quantitative information on the levels of calcium and nitrogen in the cell walls of apple (Malus domestica Borkh. cv. Cox's Orange Pippin) fruits. Samples of fruit cortex were prepared for EELS by high-pressure freezing and molecular distillation drying to minimize loss and redistribution of soluble cell wall components such as calcium. The EELS imaging successfully resolved calcium and nitrogen levels in the middle lamella and primary cell wall. When the elemental compositions of the cell walls of Cox's apples from two sites in the UK were compared at harvest or after 6 months storage, the orchard which always produced consistently firmer fruit had significantly lower levels of cell wall calcium and higher levels of cell wall nitrogen. This result was unexpected since firm texture in apples and other fruits has been commonly associated with elevated levels of fruit calcium. The nitrogen-rich material in the sections used for EELS was insoluble in acidified methanol, indicating that it represented a high-molecular-weight component in the cell wall. Furthermore, total tissue hydroxyproline levels were greatest in material with elevated cell wall nitrogen, suggesting enhanced levels of wall structural proteins in the tissue. These data indicate a correlation between increased amounts of cell wall nitrogen and firm fruit texture. The possible role of cell wall proteins in determining the textural properties of fruit tissue is discussed. Received: 19 November 1998 / Accepted: 28 January 1999     

Maturation of rat dendritic cells during intrahepatic translocation evaluated using monoclonal antibodies and electron microscopy

Specific populations of hepatic sinusoidal cells were stained with monoclonal antibodies that recognize monocytes/macrophages (ED1), tissue macrophages (Kupffer cells) (ED2), MHC class II (Ia) antigen (MRC OX6), and dendritic cells/γ,δ T-cells (MRC OX62) and analyzed by light and electron microscopy. The majority of ED1⁺ and/or ED2⁺ cells were localized to the hepatic parenchyma, whereas OX6⁺ and/or OX62⁺ cells were more densely distributed within Glisson’s sheath than in the hepatic parenchyma. Double-immunoperoxidase staining of normal liver for ED1, ED2, and OX6 identified dendritic cells (DC) of two different phenotypes, ED1⁺ED2^–OX6⁺ and ED1^–ED2^–OX6⁺. DC can be classified into three different types based on ultrastructural characteristics. The first type (type I) is characterized by one or more long cytoplasmic processes and a well-developed lysosomal system. The second type (type II) has an inconspicuous lysosomal system, abundant hyaloplasm, and characteristic short cytoplasmic processes. The third type (type I–II) has cytologic features intermediate between those of type I and type II DC. At the electron-microscopic level, these three cell types are found in the sinusoidal lumen, whereas the majority of type II DC are located in the space of Disse and Glisson’s sheath. Furthermore, some OX6-labeled elongated DC appeared to traverse the lumen of sinusoids through endothelial pores to enter the space of Disse. One hour after intravenous injection of latex particles (0.81 μm in diameter), numerous latex-laden dendritic cells (ED1⁺OX6⁺, type I and type I–II) were detected in the lumen of hepatic sinusoids, but not in the space of Disse or Glisson’s sheath. These findings suggest that normal rat liver contains resident dendritic cells which downregulate phagocytic activity and mature into potent accessory cells during migration from the portal vein toward the central vein. These DC then traverse the sinusoidal lumen to the hepatic lymph system via the space of Disse. Received: 8 May 1998 / Accepted: 15 June 1998     

Reprint of "Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)" [J. Struct. Biol. 160 (2007) 35-48

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides 3-D distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2-D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L_2,3 edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell’s nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.     

Cathodoluminescence-emitting lipid droplets in rat testis: a study by analytical color fluorescence electron microscopy

Cathodoluminescence (CL) from lipid droplets (LDs) in the rat testis was examined by analytical color fluorescence electron microscopy. The results show that (1) the Cl at wavelengths of 320 nm (CL320) and 450 nm (CL450) is derived from cholesterol esters and a mixture of lipids including vitamin A esters, respectively; (2) CL320 in the LDs of Leydig cells sharply decreases on postnatal day 21, while CL320 and CL450 in the LDs of Sertoli cells begin to be detectable; (3) the CL450-emitting LDs in seminiferous tubules, whose distributional patterns display cyclic changes during the spermatogenic cycle, are involved in spermatogenesis; and (4) the intensity of CL as well as the distributional patterns of CL-emitting LDs in testicular cells change after hypophysectomy, vitamin-A deficiency, and treatment with ethylene dimethane sulfonate and testosterone propionate. This study demonstrates that analytical color fluorescence electron microscopy is a useful tool for in-vivo observation of some specific compounds which cannot be visualized by other methods.     

Environmental modulation of calcium and phosphorus metabolism in embryonic snapping turtles (Chelydra serpentina)

Summary Eggs of common snapping turtles (Chelydra serpentina) were incubated on wet (–150 kPa water potential) and dry (–950 kPa) substrates in a laboratory study assessing the effects of the hydric environment on patterns of mobilization of calcium and phosphorus by developing embryos. We found that embryos developing in wet environments withdrew nutrients from their yolk faster, grew more rapidly, and incubated longer than embryos exposed to dry environments. Turtles developing in both environments absorbed calcium from the yolk at similar rates and depleted the yolk of almost its entire reserve of calcium prior to hatching. Calcium withdrawn from the yolk was supplemented with calcium mobilized from the eggshell, but embryos in wet environments obtained substantially more calcium from the eggshell than did those in dry settings. Embryos obtained all of the phosphorus used in skeletogenesis from the yolk, but those incubating in wet environments mobilized phosphorus from this compartment more rapidly than did those in dry settings. Exposing embryonic snapping turtles to wet environments apparently allows them to make more efficient use of the transitory source of calcium in the eggshell than is possible in dry environments. However, the residual yolk in hatchlings from both wet and dry environments contains too little calcium to support the growth of hard and soft tissues in neonates at rates similar to those characterizing the growth phase of development in embryogenesis.     

Nature of micellar calcium phosphate in cows' milk as studied by high-resolution electron microscopy

The nature of the inorganic calcium phosphate in the casein micelle of cows' milk has been studied by high-resolution electron microscopy. No periodic lattice spacings could be imaged, and diffraction patterns were of the diffuse amorphous type. Short-range order of less than 15 Å may be present, but the results indicate that there is no long-range order in micellar calcium phosphate.     

The relationship between odontoblasts and pulp capillaries in the process of enamel- and cementum-related dentin formation in rat incisors

Summary The relationship between odontoblasts and pulp capillaries in the process of dentinogenesis was studied in rat lower incisors, both on the labial and lingual sides, using light and transmission electron microscopy. The odontoblasts showed remarkable differences from the apical to the incisal end. Near the apical end of the tooth, immature odontoblasts, which were thought to be involved in the formation of the mantle dentin, were arranged in a single layer, and continuous capillaries were located just beneath the odontoblasts. In the middle of the tooth, mature odontoblasts with highly developed cell organelles and notable processes formed a pseudostratified layer; fenestrated capillaries were found between these cells close to the predentin. The height of the odontoblast layer and the rate of dentin deposition on the labial (enamel-related) side was significantly greater than that on the lingual (cementum-related) side. Near the incisal end, cementum-related odontoblasts gradually decreased in height and number to become post-odontoblasts that produced atubular dentin; continuous capillaries were located subjacent to the post-odontoblasts. On the labial (enamel-related) side, however, odontoblasts retained their pseudostratification; fenestrated capillaries were still observed in the odontoblast layer. No atubular dentin was formed on the labial side.     

Immunolocalization of proteoglycans and bone-related noncollagenous glycoproteins in developing acellular cementum of rat molars

To elucidate the roles of proteoglycans (PGs), bone sialoprotein (BSP), and osteopontin (OPN) in cementogenesis, their distribution was investigated in developing and established acellular cementum of rat molars by an immunoperoxidase method. To characterize PGs, antibodies against five species of glycosaminoglycans (GAGs), chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), unsulfated chondroitin (C0S), dermatan sulfate (DS), and keratan sulfate (KS) were used. Routine histological staining was also applied. With onset of dentin mineralization, the initial cementum appeared on the dentin surface as a hematoxylin-stained fibril-poor layer. Subsequently, primitive principal fibers attached to the initial cementum. As the acellular cementum containing extrinsic fibers covered the initial cementum, the initial cementum formed the cemento-dentinal junction. Following immunohistochemistry at the earliest time of cementogenesis, the initial cementum was intensely immunoreactive for C4S, C6S, C0S, BSP, and OPN. After the initial cementum was embedded, neither the cemento-dentinal junction nor the cementum was immunoreactive for any GAG species. However, the cementum and cemento-dentinal junction were consistently immunoreactive for BSP. Although the cemento-dentinal junction was consistently immunoreactive for OPN, the remaining cementum showed no significant immunoreactivity. Thus, initial acellular cementogenesis requires a dense accumulation of PGs, BSP, and OPN, which may be associated with the mineralization process independently of collagen fibrils and initial principal fiber attachment.     

An immunocytochemical study of pulpal responses to cavity preparation by laser ablation in rat molars by using antibodies to heat shock protein (Hsp) 25 and class II MHC antigen

Initial responses of odontoblasts and immunocompetent cells to cavity preparation by laser ablation were investigated in rat molars. In untreated control teeth, intense heat shock protein (Hsp) 25 immunoreactivity was found in the cell bodies of odontoblasts, whereas cells immunopositive for the class II major histocompatibility complex (MHC) antigen were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused the destruction of the odontoblast layer and the shift of most class-II-MHC-positive cells from the pulp-dentin border toward the pulp core at the affected site. Twelve hours after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, but subsequently disappeared from the pulp-dentin border together with Hsp-25-immunopositive cells by 24 h after the operation. By 3–5 days postoperation, distinct abscess formation consisting of polymorphonuclear leukocytes was found in the dental pulp. The penetration of masses of oral bacteria was recognizable in the dentinal tubules beneath the prepared cavity. These findings indicate that cavity preparation by laser ablation induces remarkable inflammation by continuous bacterial infections via dentinal tubules in this experimental model, thereby delaying pulpal regeneration.This work was supported by Grant-in-Aid for Scientific Research to promote 2001-Multidisciplinary Research Projects in 2001–2005, and KAKENHI (C) (nos. 12671765 and 14571727 to H.O.) from MEXT     

Patterns of mobilization of calcium,magnesium, and phosphorus by embryonic yellow-headed blackbirds(Xanthocephalus xanthocephalus)

Summary Embryonic blackbirds(Xanthocephalus xanthocephalus) obtain most of their calcium from the eggshell (85 90%), but all of their phosphorus comes from reserves in the yolk (80–85%) and albumen (15–20%). Approximately equal amounts of magnesium are supplied by the eggshell, the yolk, and the albumen. Yolk is depleted of magnesium and phosphorus during embryogenesis, but excess calcium absorbed from the eggeshell is stored in the yolk. Consequently reserves of calcium in the yolk actually increase 8-fold during embryonic development. Our results reveal that altricial birds manifest patterns of mobilization and deposition of calcium and other elements similar to those described for precocial species. Evolution of altriciality from precocity evidently did not entail major changes in how embryonic birds meet the challenge of obtaining the calcium, magnesium, and phosphorus required for development.     

Effect of phlebotomy on the ferritin iron content in the rat liver as determined morphometrically with the use of electron energy loss spectroscopy

Summary Phlebotomy of untreated and iron-loaded rats results in a significant decrease in total liver iron. In ironloaded rats a marked decrease in iron-containing particles is observed ultrastructurally in lysosomes and cytoplasm of hepatic sinusoidal cells but not in parenchymal cells. This remarkable phenomenon was further investigated in a morphometric study, based on element-specific (iron) distribution images made in situ in the parenchymal cell by means of electron energy loss spectroscopy. With the use of this technique it could be shown that in spite of phlebotomy the ferritin iron content of the iron-loaded liver parenchymal cell is not decreased.     

Pituitary adenylate cyclase-activating peptide (PACAP) immunolocalization in lymphoid tissues of the rat

Pituitary adenylate cyclase activating peptide (PACAP) is a novel peptide isolated from the ovine hypothalamus. PACAP exists in 2 molecular forms with 27 (PACAP27) or 38 (PACAP38) amino acid residues. PACAP localization was studied by immunohistochemical methods in central (bone marrow and thymus) and peripheral (spleen, lymph nodes and duodenal mucosa) lymphoid tissues with antisera raised against PACAP27 or PACAP38. PACAP-positive cells were found in all lymphoid tissues examined. These cells were highly positive for PACAP38 but were negative for PACAP27. Morphologically, they were small mononuclear cells with relatively scarce cytoplasm and lymphocyte-like features. PACAP38-positive cells were abundant in peripheral lymphoid tissues (i.e., mesenteric lymph nodes). In the duodenal mucosa, PACAP38-positive cells were located either in the lamina propria or epithelium. These results suggest that PACAP38-positive cells are present within lymphoid tissues and may represent a lymphocyte-like cell subpopulation that has a potential role in cell-to-cell interactions in the immune system and in the integrated communication between neuroendocrine and immune systems.