Microscopic analysis and seasonality of gemma production in the freshwater red alga Hildenbrandia angolensis (Hildenbrandiales, Rhodophyta)

The development and release of the unique vegetative propagules of the freshwater encrusting alga Hildenbrandia angolensis Welwitsch ex West et West, gemmae, were studied using several different microscopic and histochemical techniques. In addition, the seasonality of gemma production was monitored bimonthly over a 12‐month period in two spring‐fed streams in Texas, USA. Gemmae differentiate within the thallus and are subsequently released from the surface of the crust. Release of the gemmae most likely occurs by digestion of surrounding cells, as suggested by the presence of starch granules and lipid globules in the region between the released gemma and the thallus. The initial separation of the gemmae from the thallus occurs from the sides of the gemma or the bottom, or possibly simultaneously. Contrary to previous studies, we have observed that gemma production occurs endogenously within the thallus of freshwater Hildenbrandia, rather than on the surface of the crust in raised structures. Histochemical tests and electron microscopic examination indicate that the cells of the gemmae contain a large amount of floridean starch. The starch granules frequently form rings surrounding the nuclei of both gemma and thallus cells; a feature infrequently reported for florideophyte red algae. Our seasonality investigations indicate that large fluctuations in gemma production occur over 1 year, but at least some gemma production continues year‐round in the streams examined.     

X-ray microanalysis of biological specimens by high voltage electron microscopy

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200–400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory.

X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/B ratios at 300 kV.

From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300–400 kV was very useful resulting in high P/B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

X-ray micronalysis of copper and sulphur-containing granules in the fat body cells of homopteran insects

Regions of the fat body of larvae of Chaetophyes compacta and Pectinariophyes sp. (Machaerotidae, Homoptera) which are closely associated with mycetomes have been analysed by electron probe X-ray microanalysis. It is shown that cells in these regions contain electron probe X-ray microanalysis. It is shown that cells in these regions contain electron dense granules which are rich in copper and sulphur. These two elements occur in the atomic ratio of 3:2 respectively. It is conjectured that copper may be bound to a sulphur containing metallothionein and that the granules represent either the end products of copper detoxification or serve as copper stores for synthesis of enzymes and macromolecules by the mycetomal symbionts.     

X-ray microanalytical studies on cryofixed blood cells of the ascidian Phallusia mammillata

Summary Cryofixed blood morula cells of Phallusia mammillata (Cuvier), which are considered to be vanadium-accumulating cells, were examined by X-ray microanalysis using STEM (scanning transmission electron microscopy) and SEM (scanning electron microscopy). It is thought that cryopreparation preserves the native distribution of diffusible elements such as sodium, chlorine, and potassium, and prevents the displacement of vanadium, all of which may occur during conventional preparation. The results show that morula cell globules contain a large amount of sulphur and chlorine, and some sodium, magnesium, bromium and potassium, but very little or no vanadium.     

The distribution of zinc, cadmium, lead and copper within the hepatopancreas of a woodlouse

The distribution of metals within the hepatopancreas of Oniscus asellus (Crustacea, Isopoda) from two uncontaminated sites, and two sites contaminated with zinc, cadmium and lead, has been studied by atomic absorption spectrophotometry, light microscopy, transmission and scanning electron microscopy and X-ray microanalysis. The hepatopancreas contains two types of intracellular granule. The first type, in the S cells, are spherical granules which contain copper, sulphur and calcium. In woodlice from contaminated sites, these ‘copper’ granules, also contain zinc, cadmium and lead. The second type, in the B cells, are flocculent deposits which contain iron. In woodlice from contaminated sites, these ‘iron’ granules also contain zinc and lead. Moribund woodlice from contaminated sites have large numbers of ‘copper’ and ‘iron’ granules in the hepatopancreas and a fine deposit of zinc and lead on the membranes of the cells. There are numerous microorganisms in close association with the microvillous border of the hepatopancreas of woodlice from all four sites. Within the microorganisms of Oniscus asellus from contaminated sites, there are deposits of material which contain zinc, lead, calcium and phosphorus ‘Copper’ and ‘iron’ granules could have evolved as storage sites for essential metals to be utilized when demand from the body exceeds uptake from the food. Woodlice in contaminated sites may be able to ‘detoxify’ potentially harmful amounts of essential and non-essential metals by storing them in a relatively insoluble form within these granules.     

X-ray microanalysis of freeze-dried digestive mucus in Anguilla anguilla L: modifications of ion content during the early steps of secretion

Summary— Digestive mucus of sea-water adapted eels has been observed and analyzed by the scanning electron microscope (SEM) after rapid freezing at liquid nitrogen temperature followed by freeze-drying. No chemical procedures were used in this technique. This allowed the maintenance of the mucous coating. Preliminary X-ray microanalysis carried out on freeze-fractured and freeze-dried samples of the oesophagus showed a decrease of K⁺ and an increase of Ca²⁺ and Cl^? from the basal part of the mucous cell towards its the apical part. This technique has proven to be satisfactory for it prevents translocation and loss of diffusible elements in situ and allows X-ray microanalysis in the SEM.     

Cadmium uptake,localization and detoxification in <Emphasis Type="Italic">Zea mays</Emphasis>

Cadmium uptake, translocation and localization in maize roots and shoots at the tissue and cellular level were investigated. Metal accumulation in plant organs as well as symptoms of Cd toxicity were closely correlated with an increase in Cd concentration applied (5 – 300 M). Most of the metal taken up was retained in roots, mainly inside the cells of endodermis, pericycle and central cylinder parenchyma. Accumulation of phytochelatins and related peptides also depended on Cd concentration in the nutrient solution.     

Characterization of the cellular response during apoptosis induction in cadmium-treated hep G2 human hepatoma cells

Cadmium is a toxic transition heavy metal of continuing occupational and environmental concern, with a wide variety of adverse effects on regulation of gene expression and cellular signal transduction pathways. Injury to cells by cadmium leads to a complex series of events that can culminate in the death of the cell. It has been reported that cadmium induces apoptosis in many cell lines. However, the morphological characteristics leading to apoptosis or subsequent regeneration in cells exposed to cadmium have not been clarified. We evaluated whether human hepatoma cells maintained in culture undergo apoptosis when exposed to cadmium. Cytotoxic activity of cadmium on Hep G2 cells determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. A DNA ladder assay was performed by electrophoresis. Cell cycle analysis was quantified by flow cytometry. Nuclear morphology was studied by fluorescence microscopy after staining with propidium iodide and Hoechst 33342. Morphologic alterations in culture hepatocytes treated with CdCl₂ were observed by transmission electron microscopy. We have demonstrated that apoptosis is a major mode of elimination of damaged HepG2 cells in cadmium toxicity and it precedes necrosis.     

Cellular cadmium distribution in the common winkle, Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Various tissues of common winkles, Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

Cellular cadmium distribution in the common winkle,Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Summary Various tissues of common winkles,Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

BROMINE IN THE CUTICLE OF POLYSIPHONIA NIGRESCENS: LOCALIZATION AND CONTENT1

The cuticles and chloroplasts of young and old fronds of Polysiphonia nigrescens (Huds.) Grev. were analysed by quantitative X-ray microanalysis for their content of bromine. Cuticles of old fronds contained 1.7 ± 0.14% bromine while those of young ones contained only 0.94 ± 0.22% bromine. The bromine content of the chloroplasts showed considerable variations between individual chloroplasts, but was found not to increase with age. Young fronds contained four separate layers in the cuticle when observed under the electron microscope. In the old fronds three of these layers disappeared while the remaining one became thick and very electron dense.     

Sub-cellular localization of Ni in the hyperaccumulator, Hybanthus floribundus (Lindley) F. Muell

The cellular and subcellular distribution of Ni within leaves of Hybanthus floribundus (Lindley) F. Muell, a hyperaccumulator of Ni, was investigated at relatively high spatial resolution using energy‐dispersive X‐ray microanalysis (EDAX). Elemental distribution maps showed that Ni was predominantly localized in the vacuoles of epidermal cells in the leaves. Quantification of Ni revealed concentrations up to 275 mmol kg^?1 (embedded tissue) in some epidermal vacuoles. The accumulation of Ni in these cells was associated with a decrease in the concentration of Na and K. There was no indication that Ni was associated with P, S or Cl in the vacuoles. Ni was also concentrated on the outside of cell walls throughout the leaves, indicating that apoplastic compartmentation is also involved in Ni tolerance and accumulation in this plant.     

Cytochemical and X-ray microanalytical studies of calcium in the extending zone, particularly in the rhizodermis of Zea mays roots

Calcium was detected in CaCl (10 m M )-pretreated roots of Zea mays L. (cv. LG 11) by means of electron probe X-ray microanalysis with energy-dispersive spectrometry (EDS) using embedded samples, fixed by the antimonate staining procedure. A high level of calcium was found where large amounts of antimonate precipitates were observed by light or transmission electron microscopy. In the elongation zone, after 20 h in humid air following a 2 h CaCl₂ pretreatment, the level of calcium was higher in trichoblasts than in atrichoblasts. In these cells it was detected mainly in the walls and nucleus, and antimonate staining was observed in the walls. Abundant precipitates containing calcium were associated with the nucleus, vacuoles, mitochondria and endoplasmic reticulum of non-differentiated cells, whereas they were confined to the walls of these cells just after the CaCl₂ pretreatment. The involvement of calcium in the formation of root hairs is discussed.     

Studies on cadmium localization in the water fern Azolla

Uptake of cadmium by the water fern Azolla filiculoides (Lamarck) was characterized, and the localization of the metal in the roots and shoots as well as its effects on some essential elements were studied. Cadmium was accumulated at a rapid rate in Azolla plants within 1 h and then more gradually up to 77 h. Cadmium content was increased in the inner epidermis, cortex and bundle cell walls of the root between 33 and 77 h. This accumulation was characterized by the appearance of small dark grains with high content of cadmium, phosphate and calcium along the epidermal cells. The shoots of Azolla plants grown in the presence of cadmium showed high content of cadmium only in the ventral lobe submerged in the growth medium, suggesting that cadmium was absorbed directly from the medium. The effect of cadmium on essential elements in Azolla was characterized by simultaneous increases in calcium and cadmium content in the root and shoot and by decreases in magnesium content in the root and potassium content in the shoot.     

Ultrastructural Localization of Calcium in the Presumptive Ectodermal Cells in Gastrulae of the Newt, Cynops pyrrhogaster, as Revealed by Cytochemistry and X-Ray Microanalysis

The ultrastructural localization of calcium in the presumptive ectodermal cells of gastrulae of the newt, Cynops pyrrhogaster , was examined by cytochemical methods and X-ray microanalysis (XMA). The cells were fixed with solutions that contained potassium oxalate, potassium ferricyanide and potassium pyroantimonate to preserve the localization of intracellular calcium in situ and for the analysis of electron density due to calcium. Electron-dense deposits associated with the localization of calcium were observed under the electron microscope. Specificially, pigment granules, round vacuoles, endoplasmic reticulum and mitochondria as well as the extracellular matrix were observed to contain calcium. In addition, XMA clearly demonstrated the localization of calcium in all of these electron-dense organelles and yolk granules.     

X-ray microanalysis of black piedra

The elements present in the fungal structures produced by Piedraia hortae in vivo and in vitro have been investigated using electron microscopy X-ray microanalysis. Phosphorus, sulphur and calcium were detected in the nodules which developed on hair and on colonies on culture. These elements belong to the extracellular material that compacts the pseudoparenchymatous organization of the fungus. They may be present due to the capacity of melanin-like pigments to sequester ions and/or they may form part of the sulphates and phosphates of the polyanionic mucopolysaccharides that constitute the extracellular material. Environmental contaminants such as aluminium, silicon and iron were detected exclusively on the surface of the nodule. They were deposited or linked to the residual molecules produced during the breakdown of the cuticular keratin. The advantages of these techniques for elucidating the chemical nature of fungal structures are discussed.     

Studies on the distribution of Na and CI in two species of lupin (Lupinus luteus and Lupinus angustifolius) differing in salt tolerance

The distribution of K, Na and CI in various tissues was studied in two species of lupin, Lupinus luteus L. (ex. Portugal) and L. angustifolius L. (cv. Kubesa) under conditions of NaCl-induced salinity stress. L. luteus appeared more tolerant to salt and less effective in excluding Na and Cl from its above-ground parts than L. angustifolius . Electron probe X-ray microanalysis of vacuolar contents of individual root cells of salt-treated L. luteus showed a decreasing gradient of Na and CI contents from the epidermis inwards, but in the inner cortical cells adjacent to the pericycle/endodermis, Na levels were again high while Cl remained low. L. luteus may be inefficient in restricting entry of Na into the shoot because of a limited capacity for Na storage in the vacuoles of root cells. In addition, this species appears to exclude Cl from the vacuoles of inner cortical root cells but not from its symplastic pathway. In the leaves of L. luteus , Cl levels were particularly high towards the lower surface (abaxial side), while Na accumulated in the adaxial side of the leaf. Likewise, different cells of the petiole appeared to place unequal demands on Na and Cl for osmotic adjustment. It is suggested that excess Na in the adaxial side of the leaf and the generally high levels of Na and CI in the foliage may explain the 50% fresh weight stimulation which was observed in L. luteus but not in L. angustifolius at 50 m M NaCl. However, the halophytic response of L. luteus is limited to moderate concentrations of NaCl since 100 m M NaCl caused severe necrosis and leaf abscission.     

Heavy metals in the terrestrial isopod Porcellio scaber latreille. I. Histochemical and ultrastructural characterization of metal-containing lysosomes

Different populations of metal-loaded and uncontaminated Porcelio scaber Latreille were studied. Combined light and electron microscopial methods as well as X-ray microanalysis were applied for localization and characterization of intracellular sites of metal deposition within the small cells of hepatopancreas. By means of cytochemistry and X-ray microanalysis it was shown that membrane-limited vesicles are important sites of deposition for lead, copper, zinc and probably smaller amounts of cadmium. The vesicles also contained phosphorus. They are identical with the reported cuprosomes and belong to the lysosomal system. In addition, considerable amounts of lead, copper, and cadmium were found in small structures outside of membrane-limited organelles.Abbreviations EDX energy-dispersive X-ray - P. scaber Porcellio scaber - STEM scanning transmission electron microscope - TEM transmission electron microscope - v volume - wt weight     

Si在燕麦籽粒中的富集及其与其它8种元素的关系

通过环境扫描电镜结合X射线电子探针显微分析技术,对22个基因型的燕麦籽粒皮层、糊粉层、近糊粉层和颖果中部的Si含量进行测定.结果表明,Si元素在燕麦籽粒不同部位的含量有明显差异,皮层和糊粉层Si含量较高,近糊粉层和颖果中部含量较低,在颖果中Si主要富集在糊粉层中;而且不同基因型燕麦籽粒同一部位或不同部位Si的积累量也有较大差异,这可能是由遗传差异引起的.糊粉层中的Si含量影响着近糊粉层和颖果中部的Si含量.另外,籽粒不同部位Si含量与P、Ca、Mg、S、Al、Pb含量之间存在显著或极显著的非线性关系,颖果中部的Si含量与K、Cd含量之间不存在显著的非线性关系.说明Si在燕麦籽粒中富集的同时也影响着P、Ca、Mg、S、Al、Pb等元素的富集.