Microanalysis and image processing of stable and radioactive elements in ecotoxicology. Current developments using SIMS microscope and electron microprobe

Ecotoxicological investigations were performed on two sets of biological models. The first one concerns marine pollution and was composed of invertebrates (molluscs and crustaceans) contaminated by stable or radioactive elements originating from wastes discharged into sea water. The second one concerns freshwater pollution and was composed of vertebrates (fish) contaminated by aluminium which was dissolved in rivers, as a consequence of an atmospheric pollution by acid rain. Mechanisms involved in the uptake, storage and elimination processes of these toxicants were studied, with a special emphasis on cellular and subcellular aspects of concentration sites. Two microanalytical methods were employed: secondary ion mass spectrometry (SIMS), using the ion microscope and the ion microprobe, and X-ray spectrometry using the electron microprobe (EMP). SIMS, which enables the visualization of trace elements, was associated with an image processing system using a highly sensitive television camera connected to an image computer. Polychromatic images were obtained, allowing to establish the cellular distribution of metal contaminants. In marine organisms, the target organs and tissues of Al, rare earth elements (Tm and La) and radionuclides (U, Pu, Am) were shown to be mainly digestive gland and exoskeleton. The target organelles were shown to be spherocrystals and lysosomes where the enzymatic lysosomal coprecipitation with phosphorus was observed. Amoebocytes, which are enzymatically equipped with lysosomal phosphatase, were involved in the phagocytic clearance of metal pollutants. In trout, two processes appeared to be involved in Al accumulation. The first one corresponds to the well known insolubilisation of Al phosphate, within lysosomes of organs devoted to uptake and excretion such as gill and kidney. The second one demonstrates that organs and tissues which cannot eliminate, such as bone, heart and brain, retain Al, exhibiting a high intracellular metal concentration; moreover, large Al deposits inducing nervous tissue destruction have been observed. Data have been discussed in connection with the relationship between man and his environment.     

Biosorption Potency of Aspergillus niger for Removal of Chromium (VI)

Aspergillus niger isolated from soil and effluent of leather tanning mills had higher activity to remove chromium. The potency of Aspergillus niger was evaluated in shake flask culture by absorption of chromium at pH 6 and temperature 30°C. The results of the study indicated removal of more than 75% chromium by Aspergillus niger determined by diphenylcarbazide colorimetric assay and atomic absorption spectrophotometry after 7 days. Study of microbial Cr(VI) reduction and identification of reduction intermediates has been hindered by the lack of analytical techniques that can identify the oxidation state with subcellular spatial resolution. Therefore, removal of chromium was further substantiated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX), which indicated an accumulation of chromium in the fungal mycelium.     

Techniques and applications of three‐dimensional electron microscopy in entomological research

Structural studies using two‐dimensional (2D) images show limitations in understanding the structure and functions of cellular organelle and protein. To overcome the difficulty, over the last few years 3D reconstruction techniques using electron microscopy have been developed at extremely high speed. In this paper, currently available 3D reconstruction techniques of electron microscopy (such as electron tomography, serial section analysis and single particle analysis) are introduced using our data as examples of the application. The 3D structure of mitochondria with the defect of mitochondrial protein in round worm, Caenorhabditis elegans, through electron tomography, the cell–cell interaction in lamina of Drosophila melanogaster by serial‐section using ultramicrotome and high‐voltage electron microscopy and a thin filament related to muscle contraction in Drosophila melanogaster were used for examples of the application. These results through 3D reconstruction reveal the structural changes in a cellular organelle and protein that had not been shown by 2D structure.     

Lysozyme expression in the rat parotid gland: light and electron microscopic immunogold studies

 Lysozyme (muramidase) is capable of direct bacteriolytic action by hydrolyzing glycosidic bonds in bacterial cell walls. Although it is broadly distributed in vertebrate tissues and secretions, the cellular and subcellular localizations of the enzyme are still not well known. The present study examines the distribution of lysozyme expression in the various cell types of LR gold-embedded rat parotid gland, applying a postembedding immunogold-silver staining technique for light microscopy. Simultaneously, a postembedding immunogold method for electron microscopy was used to determine the cellular compartments engaged in the biosynthesis and exocytosis of lysozyme. Silver-amplified immunogold staining for lysozyme demonstrated identical localization in both paraffin and semithin LR-gold sections: in the supranuclear parts of acinar and intercalated duct cells. Staining intensity varied even between adjacent cells. In the electron microscope, immunogold labeling was detected over the cell compartments associated with protein synthesis and exocytosis in acinar and intercalated duct cells. Lysozyme antigenic sites were visible over endoplasmic reticulum and throughout the Golgi apparatus, being intense over the trans-Golgi network, but even stronger in the condensing vacuoles and most prominent over secretory granules in both cell types. The findings provide the first immunocytochemical evidence of the synthesis and secretion of lysozyme in parotid acinar and intercalated duct cells. Accepted: 3 December 1996     

Light and electron microscope localization of beta-glucuronidase activity in the stomach and digestive gland of the marine gastropod Littorina littorea

Summary An azo dye technique was used to investigate localization of the acid hydrolase,-glucuronidase, at light and electron microscope level in the stomach and digestive gland of the marine periwinkleLittorina littorea. Activity for-glucuronidase was located principally within digestive cells of the digestive gland and also associated with the microvillous border and epithelial cells lining the stomach. At the light microscope level all digestive tubules showed activity which appeared essentially restricted to the large heterolysosomes of the digestive cells. However not all digestive cells showed activity. In the electron microscope, reaction product was apparent in all types of macrovesicle in the digestive cells although not all stained positively. Heterophagosomes typically showed reaction product around their periphery or associated with the electron opaque contents. Activity was commonly seen around the apical edge of heterolysosomes where merging of heterophagosomes into heterolysosomes was apparent. Reaction product was commonly located within small electron lucent vesicles which lined the internal membrane of the heterolysosomes but sometimes also associated with flocculent, electron opaque contents. In the stomach dense clusters of reaction product were visible in lysosomes in the basal region of the epithelial cells and in the large granular inclusions of the secretory cells.     

A histological and ultrastructural study of the cells of the mantle edge of a marine bivalve, Cerastoderma edule

The cells of the mantle edge of Cerastoderma edule are described after light and electron microscopical observations. Histochemical tests for calcium in the mantle edge and digestive gland (Dahl, 1952; McGee-Russell, 1958) and analytical electron microscopy of the mantle edge of C. edule both failed to show calcium. Similar results were obtained for Mytilus edulis and Chlamys opercularis. However, calcium was detected in the digestive gland of the terrestrial gastropod Helix aspersa. The outer secretory fold of the mantle edge is composed of tall columnar cells. These cells have highly convoluted lateral cell membranes with which many mitochondria are closely associated. These features are indicative of an ion pump which could move calcium from the mantle space to the extrapallial cavity (compare with Bubel's findings, 1973b). There are many features of the cells lining the periostracal groove of C. edule that have not been reported previously (e.g. Bubel, 1973b) and which are now discussed. The periostracal sheet arises within a line of basal cells in the fundus of the periostracal groove. Within these cells the periostracum in section has a spiral form. It is suggested that the newly formed periostracum adheres to the microvillous border through secretions produced from the middle fold cells lining the groove. During its passage along the groove the periostracum is gradually thickened by secretions from the outer fold cells.     

FTIR spectroscopy and scanning electron microscopic analysis of pretreated biosorbent to observe the effect on Cr (VI) remediation

Various chemical and physical treatments have been applied to indigenously isolated cyanobacterial strain, Lyngbya putealis HH-15, to observe the effect on chromium removal capacity. Pretreatment with hydrochloric acid (99.1%) and nitric acid (98.5%) resulted in enhanced chromium removal as compared to untreated control biosorbent (98.1%). Pretreatment with acetic acid (97.9%), methanol (97.0%), calcium chloride (96.0%), hot water (95.2%), and sodium hydroxide (93.9%) did not improve the chromium removal capacity of biosorbent. Fourier transform infrared spectrometry (FTIR) and scanning electron microscopy (SEM) analysis identified changes in biomass functionality and availability after physical and chemical modification—the results of which were in agreement with metal removal studies. In conclusion, this acid-treated biosorbent represents a suitable candidate to replace conventional removal technologies for metal-bearing wastewaters.     

Competing ion effect on chromium adsorption with fresh and starved subsurface bacterial consortium

Summary The sorption of chromium (Cr⁺⁶) was investigated for 50 day starved and fresh cells in the presence of sulfate ion as a competing ion at three pH values (pH 6.0, 7.5 and 9.0). The chromium distribution in the subcellular components was also determined by conducting cell fractionation. Although a previous study showed that cells which had been starved for 50 days prior to being exposed to a chromium containing solution sorbed 10–15% more chromium than did fresh cells (Kong et al., 1992a), this study demonstrated that such cells did not sorb more chromium in the presence of sulfate ion.     

Ultrastructure and histochemistry of the foregut in<Emphasis Type="Italic"> Wirenia argentea</Emphasis> and<Emphasis Type="Italic"> Genitoconia rosea</Emphasis> (Mollusca,Solenogastres)

Wirenia argentea and Genitoconia rosea feed on Cnidaria like most representatives of the molluscan taxon Solenogastres (Aplacophora, Neomeniomorpha sensu Scheltema). The structure and histochemistry of the foregut are described based on histologic, semithin, and ultrathin section series. The ultrastructure was analyzed by means of transmission electron microscopy. There are two sets of unicellular glands: a narrow row of preoral gland cells opening to the preoral area, and pharyngeal gland cells in high numbers. Preoral gland cells produce serous secretions in W. argentea, but mucosubstances in G. rosea, whereas pharyngeal gland cells are similar in structure and histochemistry in both species. Based on the size and electron density of gland vesicles, five distinct types of pharyngeal gland cells can be defined. In contrast to earlier assumptions, all types of pharyngeal gland cells produce serous secretions, most probably representing digestive ferments, but no mucosubstances.     

Quantitative assessment of cell damagein situ: Electron microprobe X-ray analysis of model organisms treated with noxious species

The existing set of methods for assessing toxicity of noxas, based on experiments with whole animals (subclinical toxicity, toxicokinetics, carcinogenity, teratogenity, neurotoxicologyetc.) does not provide much information about cellular and subcellular effects such compounds may exert. We suggest to complement the current methodology by combining a traditional morphological observation in an electron microscope with a spectroscopic method of electron microprobe X-ray analysis (or X-ray microanalysis). The latter makes it possible to measure concentrations of chemical elements in invidividual cells and organelles and effects of noxas can thus be assessed (i) at subcellular level, (ii) directlyin situ and (iii) quantitatively. Concentrations of biologically important elements such as phosphorus, sulfur or zinc were measured in individual organelles in both intact and noxa-treated tissues, thus offering a possibility of comparing the effects of various noxious species at subcellular level (with the noxa previously applied to whole tissue or animal). The suggested correlation of analytical and morphological information may also provide new insights into cellular targeting of noxas (and potentially also drugs) as some organelles appear to be much more susceptible to damage than others. Presented at the 4th Mini-Symposium on Biosorption and Microbial Degradation, Prague, Czech Republic, November 26–29, 1996.     

Implementation of subcellular water mapping by electron energy loss spectroscopy in a medium-voltage scanning transmission electron microscope

The water concentration in biological cells plays a predominant role in cellular life. Using electron energy loss spectroscopy, the feasibility to measure the water content in cells has already been demonstrated. In this paper, we present an upgrade of water measurement in hydrated cryosections by spectrum imaging mode in a medium-voltage scanning transmission electron microscope. The electron energy loss spectra are recorded in spectrum imaging mode in a 2ⁿ×2ⁿ pixels array. Each spectrum is processed in order to determine the water mass content in the corresponding pixel. Then a parametric image is obtained in which grey levels are related to water concentration. In this image, it is possible to recognize the different subcellular compartments. By averaging the water concentration over the relevant pixels, we can determine the water mass content in the concerned subcellular compartment. As an example, we present water mass content measurement at subcellular level in rat hepatocytes.     

Assessment of the state of the Japanese scallopMizuhopecten yessoensis in Alekseeva Bight (Peter the Great Bay, Sea of Japan) based on morphological and biochemical parameters

The results of an integrated examination of the state of the scallopMizuhopecten yessoensis in Alekseeva Bight (Peter the Great Bay, Sea of Japan) are presented. In mollusks of different ages, shell height was measured; in animals of commercial size (over 100 mm), some size and weight characteristics (annual increment of shell and adductor muscle and soft tissue weight) were determined. The morphology of the digestive gland and gills was studied. In the adductor muscle and digestive gland, the concentration of heavy metals (HMs) (Hg, Cu, Zn, Mn, Pb, Cd, Ni, Co, and Cr) was determined. In the digestive gland, metallothionein and reduced glutathione concentration was also determined, as was the activity of glutathione-dependent enzymes (glutathione peroxidase and glutathione reductase). In scallops collected outside Alekseeva Bight, the linear growth rate and adductor muscle weight were on average 1.3 and 1.7 times greater, respectively, than in those collected in the bight. In scallop organs, numerous histomorphological alterations were revealed: digestive cell vacuolization and hemocyte infiltration of the digestive gland, hyperplasia and vacuolization of the respiratory epithelium, and connective tissue hypertrophy in gill filaments. The biochemical parameters of scallops from Alekseeva Bight differed substantially from those of mollusks collected outside the bight. We conclude that one of the factors negatively affecting the state of theM. yessoensis population in Alekseeva Bight is the contamination of the bight with HMs, especially mercury. This is consistent with the results of chemical analysis of bottom sediments and tissues of two mytilid species,Modiolus kurilensis andCrenomytilus grayanus, specimens of which were collected in the bight together with the scallops [3].     

Corrective note about R* cells of the digestive gland of the shore crab Carcinus maenas

In a previous paper, we described and discussed the possible functions of calcospherite-rich cells (R^* cells) in the digestive gland of the shore crab, Carcinus maenas. We recently realised that electron micrographs in this publication presented neither typical R^* cells nor their calcium phosphate granules. Indeed, our pictures showed spermatophores (filled with typical spermatozoa) that had contamined hepatopancreatic cell suspensions. As the present study indicates, this contamination is difficult to detect by optical microscopy because unstained R^* cells closely resemble spermatophores. However, morphological differences between these cell types appear clearly when observed by electron microscopy. The present paper describes a comparative study of cell populations isolated from female digestive glands; it validates our previous results obtained with male hepatopancreas and suggests a low containation of those male cell fractions by spermatophores.     

Correlative light electron ion microscopy reveals in vivo localisation of bedaquiline in Mycobacterium tuberculosis–infected lungs

Correlative light, electron, and ion microscopy (CLEIM) offers huge potential to track the intracellular fate of antibiotics, with organelle-level resolution. However, a correlative approach that enables subcellular antibiotic visualisation in pathogen-infected tissue is lacking. Here, we developed correlative light, electron, and ion microscopy in tissue (CLEIMiT) and used it to identify the cell type–specific accumulation of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis. Using CLEIMiT, we found that the anti-tuberculosis (TB) drug bedaquiline (BDQ) is localised not only in foamy macrophages in the lungs during infection but also accumulate in polymorphonuclear (PMN) cells.

This study uses correlative light, electron and ion microscopy (CLEIM) in vivo to reveal the intracellular fate of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis, with organelle-level resolution.     

MALDI-MS and NanoSIMS imaging techniques to study cnidarian–dinoflagellate symbioses

Cnidarian–dinoflagellate photosynthetic symbioses are fundamental to biologically diverse and productive coral reef ecosystems. The hallmark of this symbiotic relationship is the ability of dinoflagellate symbionts to supply their cnidarian host with a wide range of nutrients. Many aspects of this association nevertheless remain poorly characterized, including the exact identity of the transferred metabolic compounds, the mechanisms that control their exchange across the host–symbiont interface, and the precise subcellular fate of the translocated materials in cnidarian tissues. This lack of knowledge is mainly attributed to difficulties in investigating such metabolic interactions both in situ, i.e. on intact symbiotic associations, and at high spatial resolution. To address these issues, we illustrate the application of two in situ and high spatial resolution molecular and ion imaging techniques–matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and the nano-scale secondary-ion mass spectrometry (NanoSIMS) ion microprobe. These imaging techniques provide important new opportunities for the detailed investigation of many aspects of cnidarian–dinoflagellate associations, including the dynamics of cellular interactions.     

Mass spectrometry imaging and profiling of single cells

Mass spectrometry imaging and profiling of individual cells and subcellular structures provide unique analytical capabilities for biological and biomedical research, including determination of the biochemical heterogeneity of cellular populations and intracellular localization of pharmaceuticals. Two mass spectrometry technologies-secondary ion mass spectrometry (SIMS) and matrix assisted laser desorption/ionization mass spectrometry (MALDI MS)-are most often used in micro-bioanalytical investigations. Recent advances in ion probe technologies have increased the dynamic range and sensitivity of analyte detection by SIMS, allowing two- and three-dimensional localization of analytes in a variety of cells. SIMS operating in the mass spectrometry imaging (MSI) mode can routinely reach spatial resolutions at the submicron level; therefore, it is frequently used in studies of the chemical composition of subcellular structures. MALDI MS offers a large mass range and high sensitivity of analyte detection. It has been successfully applied in a variety of single-cell and organelle profiling studies. Innovative instrumentation such as scanning microprobe MALDI and mass microscope spectrometers enables new subcellular MSI measurements. Other approaches for MS-based chemical imaging and profiling include those based on near-field laser ablation and inductively-coupled plasma MS analysis, which offer complementary capabilities for subcellular chemical imaging and profiling.     

Structure and possible functions of the calcospherite-rich cells (R* cells) in the digestive gland of the shore crab CArcinus maenas

Summary R^*-cells of the digestive gland of Carcinus maenas have been investigated functionally and morphologically. A comparison of the capacity of separated cell suspensions to synthesize glycogen gave support to the hypothesis that R and R^* cells belong to the same cell line. The unexpected observation of R^* cells in gastric juice suggests that their release could represent a mode of redistribution of carbohydrate stores when the feeding activity of the crab is lower. Under electron microscopy, the calcospherites of R^* cells appeared to be surrounded by multiple membranous layers, and displayed tubular and vesicular structures in their core. High glucose-6-phosphatase (G6Pase) activity in the subcellular fraction that is enriched in calcospherites suggests that these membranes are derived from the endoplasmic reticulum, via a process in which the enzyme plays a key role. We propose that this is the way by which the R cell differentiates into R^* cell.     

Assessment of the adaptation capabilities of the bivalves Modiolus modiolus (Linnaeus, 1758) and Crenomytilus grayanus (Dunker, 1853) under increased levels of heavy metals in the environment

The active biomonitoring method was used to examine the changes in heavy-metal (Fe, Zn, Cu, Cd, Mn, Pb, and Ni) contents in the organs (muscle, gonads, mantle, gills, digestive gland, and kidney) of the mussels Crenomytilus grayanus and Modiolus modiolus. The dynamics of trace element concentrations in mussel organs during the experiment were compared with their subcellular distribution. The defense strategy of M. modiolus consisted of the threshold accumulation of toxic metals in all organs followed by their excretion, whereas the strategy of C. grayanus involved short-term isolation from adverse environmental influence. Under chronic pollution, in C. grayanus the main loads occurred in the digestive gland and kidney. Under acute changes in environmental conditions, the processes of regulation (detoxification/excretion) of Fe, Mn, and Pb in this species were impaired substantially.     

Ultrastructure of the crayfish antennal gland revealed by scanning and transmission electron microscopy combined with ultrasonic microdissection

The antennal gland of the crayfish Pacifasticus leniusculus was studied using standard techniques for scanning electron microscopy as well as newer procedures for ultrasonic microdissection. To clarify relationships in the nephron tubule, transmission electron microscopy was employed. The coelomosac contains elongated cells (podocytes) displaying microvilli and extensive apical blebbing. A smooth basal lamina lines the blood space that furnishes hemolymph to the coelomosac. The labyrinth consists of tall columnar cells displaying apical microvilli, numerous blebs that seem to represent an expansion of apical plasma membrane, and lateral interdigitations. The nephron tubule consists of two distinctly different areas: a proximal region of flattened cells with extensive intercellular fusions, and a distal segment of separate, dome-shaped cells. Despite many similarities between the crayfish kidney and the vertebrate nephron, there are striking differences. The amount of surface blebbing that occurs in the coelomosac and labyrinth far exceeds that of the vertebrate nephron and may reflect its importance in the function of the crayfish kidney. The cells of the coelomosac are taller than are the vertebrate podocytes and possess less obvious arms and pedicels. In addition, the proximal segment of the nephron tubule is notable for its intercellular fusions, which are not present in the vertebrate nephron. Although the function of the intercellular fusions is unknown, they may play a role in cellular communication or the redistribution of fluids or electrolytes between cells.