Occurrence, distribution, and localisation of metals in cnidarians

The Cnidaria are simple organisms that have remarkable physiological features susceptible to microscopic investigation. As a group they produce cnidae, the most complex intracellular organelles known, form symbioses with a range of unicellular algae, contain mucocytes that account for a very substantial fraction of their body mass, and form complex skeletal structures of calcium carbonate. This review summarises contributions dealing with the distribution and localisation of metals of physiological and pathological importance within soft tissues and skeletons. Whilst there have been detailed studies of microscale metal distribution, using X-ray microanalysis, in the stinging organelles or cnidocysts and in mucocytes, other cells such as symbiotic algae and the epithelial cells have received little attention. In the skeleton-producing scleractinian corals X-ray microanalysis has provided tenuous, but persistent, evidence of Ca associated with intracellular vesicles or granules in the skeletogenic epithelium, even though the investigations were technically limited. These observations may be germane to the intriguing and intransigent problem of the mechanism of coral calcification. Metal localisation in coral skeleton at the resolution of annual growth rings has been concerned with the validity of Sr/Ca and Mg/Ca ratios as thermometers for paleoclimatic studies. It is not clear whether these ratios are influenced primarily by environmental or biological parameters. Microscale analyses by X-ray microanalysis and ion microprobe indicate a much greater variability of metal ratios which suggests biological control of metal deposition. New data are provided on the elemental composition, measured by X-ray microanalysis, of cells and cell compartments in the coral Galaxea fascicularis and zooxanthellae in the anemone Aiptasia sp. New information is also presented on changing Ca/Sr ratios at the skeletal interface in Galaxea fascicularis.     

Localization of metals in cells of pterygote insects

Insects maintain the equilibrium of their internal milieu by diffusion mechanisms, but more frequently by a substantial storage of metals in the cells of numerous organs: digestive tract, Malpighian tubules, fat body, integument, and genital organs. This storage implies the precipitation, of numerous cations (Ca, Mg, K, Mn, Fe, Zn, Cu) in a structure called the spherocrystal, which originates from the endoplasmic reticulum-Golgi complex: elements precipitate on a glycosaminoglycan nucleus in thin peripheral strata. Some spherocrystals contain exclusively mineral compounds, frequently phosphates, whereas others may contain organic compounds such as urates. In some species mineralized lysosomes store Ca, Fe, Zn, and Cu. When fed additional metals found in the environment, insects such as cockroach and ant are able to stay alive and to trap the metals (Cd or Pb, for example) in the peripheral strata of spherocrystals; the cytoplasm is not altered. It seems that these insects are able to resist exposures to high levels of toxic metals. The lysosomes are able to retain toxic heavy metals (Cd or Hg, for example) within metallothionein-like proteins.     

The intracellular distribution of ions and water in rat liver and heart muscle

Local dry mass concentrations of intracellular compartments in rat heart muscle and liver cells were estimated by quantitative electron microscopy and X-ray microanalysis of ultrathin frozen-dried cryosections. The results were used to calculate elemental concentrations per litre of compartment water from the X-ray microanalytical data. Water fractions were between 80.3 ± 1.3% of wet weight in the decondensed chromatin and only 45.1 ± 1.7% in mitochondria of liver cells. The lowest water fraction in heart muscle cells was also found in mitochondria. The ionic concentrations found in the cytoplasm of liver cells and in the myofibrils are in accord with the electroneutrality rule and in osmotic equilibrium with the extracellular concentrations. The concentrations of Na, K, Cl and P both in the cytoplasm and in the regions of decondensed chromatin within the nuclei were found to be equal. However, in regions of condensed chromatin K⁺ concentrations were found to be much higher than expected for a Donnan distribution of ions free in solution. Most probably the activity coefficient for K⁺ is lower in the condensed chromatin than in the decondensed or in the cytoplasm. The same holds true for the A-band as compared to the I-band in heart muscle cells. A sequestration of K⁺ was measured also in the rough endoplasmic reticulum (RER) of hepatocytes. The Cl^? concentration in mitochondria both in heart muscle and liver cells has been measured far in excess of what might be expected from a Nernstian distribution. A coupled inward Cl^? transport in mitochondria must, therefore, be assumed.     

Cellular and subcellular coherent dynamics,biological functional properties,and system-environment interaction

In this view point paper, we briefly summarize some of the clinical, biochemical and biophysical results obtained in our research on Relaxation Response. We also qualitatively describe the theoretical biophysical model that could link them. Our work points to a unified view of the human biological system activity, joining the dynamics ruling the interactions and correlations of the microscopic components to the knowledge of their specific individual properties in the effort of going beyond a purely atomistic approach.     

The measurement of water distribution in frozen specimens

There are three techniques to measure local water fractions in the cryomicroscope. First, water content may be measured by a direct analysis of oxygen in bulk samples using a windowless detector. Secondly, mass thickness may be estimated in frozen-hydrated, then frozen-dried sections. This technique offers unrivalled spatial resolution, especially if the radiation dose in the frozen-hydrated state is kept low by the use of electron scattering techniques instead of an X-ray microanalytical background determination. External water content standards can be used instead of frozen-hydrated sections and the whole analysis can even be performed exclusively on frozen-dried sections at room temperature. Thirdly, local water fractions can be evaluated from X-ray microanalytical measurements of element concentrations per mass in the frozen-hydrated and frozen-dried state. Corrections necessary for the other techniques cancel out. However, the high radiation dose required for a fully quantitative analysis excludes the use of these methods in thin or ultrathin sections.     

Cadmium distribution and effects on ultrastructureand chlorophyll status in photobionts and mycobionts of Xanthoria parietina

In this work, we tried to go deeper inside distribution and toxicity of cadmium (Cd) in the macrolichen Xanthoria parietina (L.) Th. Fr. Thalli of this species were treated with 0 (control), 4.5, 9, 18, or 36 muM Cd for 24 or 48 hours. Transmission electron microscopy, X-ray microanalysis, and electron energy loss spectroscopy were exploited to study distribution and ultrastructural effects of Cd in thalli; spectrophotometric techniques were utilized for measuring Cd effects on chlorophyll (Chl) content; light fluorescence microscopy was used to evaluate Chl autofluorescence. The highest Cd concentration caused ultrastructural alterations both in the mycobiont and in the photobiont, more severe in the latter, decreased total Chl content and progressively quenched Chl autofluorescence. Cell wall immobilization was observed in both bionts, and evidence pointing to a Cd-binding ability by the concentric bodies in the mycobiont was also obtained. Lower Cd concentrations led to slight or even no effects on thallus structures and on Chl content and autofluorescence. The results obtained suggest that: (1) among the two bionts, the algal partner appears to be more susceptible to Cd stress, probably because of the presence of delicate and sensitive components such as the chloroplast and photosynthetic pigments; (2) a concentration threshold exists for the occurrence of evident structural and functional damage in X. parietina thalli exposed to Cd.     

X-ray microanalysis of ion distribution in frozen salt/dextran droplets after freeze-substitution and embedding in anhydrous conditions

A model system using dextran droplets of different salt solutions either frozen together, or sandwiched together after freezing and then freeze-substituted, embedded and dry sectioned has been investigated by X-ray microanalysis. X-ray maps and spot measur ements taken in transects through the interface of both droplets have shown that the P, K and Ca remain well localized in their original droplet. This validates freeze-substitution as a method for localization of these elements in biological samples. Results with Na were more variable and not always explainable. Success was achieved by the use of super-dry solvents and maintenance of a dry environment at all stages. We emphasize the need to avoid water contamination not only during freeze-substitution but also during sectioning, storage and section transfer to the electron microscope.     

The current status of fixation for electron microscopy: A review

Chemical fixation of cells has been used extensively in different fields of electron microscopy. The type of fixation can be selected to maximize retention of different cell constituents where morphology is of prime interest. For techniques such as immunocytochemistry and autoradiography, a compromise between morphology and retention of antigenicity or radiolabel has to be reached. By careful choice of fixative, buffer and other physical parameters, optimum results can be obtained for both animal and plant tissues. Emphasis has been put on more recent developments in technique.     

Absorption microanalysis with a scanning soft X-ray microscope: mapping the distribution of calcium in bone

This article describes the scanning transmission X-ray microscope operated at the National Synchroton Light Source. The application of the instrument to elemental analysis is detailed. In particular, qualitative results on the calcium distribution in human skull tissue are presented.     

Preparation of cryosections with a modified Sorvall MT2B ultramicrotome and cryoattachment

The Sorvall MT2B ultramicrotome and cryoattachment were modified to extend the duration of cutting and to overcome problems in tissue preparation. Microtome and cryochamber were encased and surrounded by an atmosphere of dry nitrogen gas at room temperature. A specially designed cold block sited within the box close to the microtome enabled tissues to be trimmed at low temperatures in the dry environment. Tissues could be readily visualized both on the trimming block and in the chamber with the existing microscope modified to improve its working distance and lighting system. During transfer from storage in liquid nitrogen, through the trimming procedure, to arrival in the cryochamber, the temperature of tissues as determined using thermocouples embedded in PVP on a stub, never exceeded 140 K. Although there was a considerable standing temperature gradient within the cryochamber, the cutting environment, specimen and edge of the knife were thermally stable. Sections could be routinely cut dry and freeze-dried within the chamber, if desired.     

Localization of metals in cells of saprophagous soil arthropods (Isopoda, Diplopoda, Collembola)

This review summarizes results on the intracellular distribution of metals in cells of woodlice (Isopoda), millipedes (Diplopoda), and springtails (Collembola), which are three major groups of saprophagous arthropods contributing to the turnover of soil organic matter. Although the impact of metals and also metal pollution has inevitably been shown at levels of higher biological organization than subcellular mechanisms in these animal groups, the aim of this review is to focus exclusively on storage sites and aspects of intracellular metal metabolism. Thus, methodologically, results obtained by microscopical techniques such as histochemistry, X-ray microanalysis, energy filter transmission electron microscopy, or laser microprobe mass spectrometry were given preference. Results from atomic absorption spectrophotometry of cellular fractions were kept to a minimum. In all three taxa, the main intracellular metal storage sites are various types of "granules" which are widely distributed throughout cell types associated with the digestive system.     

Measuring the angular dependent energy distribution of backscattered electrons at variable geometry

An aluminium semisphere system with 120 points of entry and eight detection areas, assembled on a meridian covering 0.0026 steradian each, was put over a solid bulk sample (e.g., aluminium), which was mounted in the eucentric point so that the incident electron beam could be varied by a polar rotation of the sphere in steps of 11.25 degrees. The complete angular distribution of the backscattered electrons became available by a rotation in steps of 11.25 degrees azimuthally. For this particular setup, the signals from the detection areas as well as the signal from the rest of the semisphere were amplified by operational amplifiers (Burr-Brown OPA128LM). However the signal of the semisphere was not available at that time. Specimen current measurements made the total amount of electrons accessible, providing a possibility for normalization of the results and comparison with total backscattering coefficients. By use of counter voltage variable up to 10 kV inside the detection assembly, it was possible to measure an energy resolution of the backscattered electrons for each detection area at the same time. Details of the construction and calibration procedures, possible errors, and sources of systematic deviations as well as first test results are discussed.     

Suggestions for the quantitative X-ray microanalysis of thin sections of frozen-dried and embedded biological tissues

When a microregion in a thin section of frozen-dried and embedded tissue is analysed by the conventional electron-probe X-ray continuum-normalization method, the measured quantity is in mmol of element per kg of embedded specimen. As each microregion contains an unknown amount of embedding medium, this quantity generally lies indeterminately somewhere within the wide range between mmol of element per kg of hydrated tissue and mmol of element per kg of dehydrated tissue. However, if a ‘tag’ element is incorporated in the embedding medium, the contribution of the medium to the local continuum count in each probed field should be measurable, and the X-ray data may then unambiguously yield mmol of element per kg of dehydrated tissue. This result should not be affected by shrinkage on freeze-drying or by incomplete replacement of water by embedding medium. The same X-ray data can additionally provide estimates of mmol of element per unit volume, mmol of element per kg of hydrated tissue and local dry-mass fraction. However, these estimates are subject to errors due to tissue shrinkage, incomplete replacement of water and beam damage.     

The preparation of cryosections from plant tissue: An alternative method appropriate for secondary ion mass spectrometry studies of nutrient tracers and trace metals

A method involving cryostat sectioning (10 μm thickness) and freeze-drying is presented for the preparation of plant tissue for microanalytical studies. The method is well suited for semi-quantitative imaging by secondary ion mass spectrometry (SIMS) and offers significant advantages over bulk freeze-dried or freeze-substitution preparations. Segments of corn or soybean root (5 mm) are quench-frozen, embedded externally, sectioned in a cryostat (10 μm), pressed onto ultrapure Si and slowly freeze-dried. Images of these sections with secondary electron microscopy and SIMS indicated good morphological preservation. It was possible to section tissues of a wide developmental range, as well as roots varying sixfold in diameter. SIMS images are presented which demonstrate the ability to detect and localize nutrient tracers, such as Rb⁺, following brief exposures (10 min) to the intact plant. Likewise, a toxic metal (Al) was localized in root tissue after brief exposure (<1 day) of the intact plant root to micromolar external concentrations. Elemental redistribution during processing was minimal, as demonstrated most explicitly by the lack of movement of loosely bound Ca from the outer cell walls into the adjacent embedding material. Preservation of compositional differences between cellular content and cell wall was supported by a semi-quantitative treatment of SIMS images.     

Technical advances in the sectioning of dental tissue and of on‐section cross‐linked collagen detection in mineralized teeth

Immunohistochemical detection of cross‐linked fibrillar collagens in mineralized tissues is much desired for exploring the mechanisms of biomineralization in health and disease. Mineralized teeth are impossible to section when embedded in conventional media, thus limiting on‐section characterization of matrix proteins by immunohistochemistry. We hypothesized that by using an especially formulated acrylic resin suitable for mineralized dental tissues, not only sectioning of teeth would be possible, but also our recently developed immunofluorescence labeling technique would be amenable to fully calcified tissues for characterization of dentinal fibrillar collagens, which remains elusive. The hypothesis was tested on fixed rodent teeth embedded in Technovit 9100 New®. It was possible to cut thin (1 μm) sections of mineralized teeth, and immunofluorescence characterization of cross‐linked type I fibrillar collagen was selected due to its abundance in dentine. Decalcified samples of teeth embedded in paraffin wax were also used to compare immunolabeling from either method using the same immunoreagents in equivalent concentrations. In the decalcified tissue sections, type I collagen labeling in the dentine along the tubules was “patchy” and the signal in the predentine was very weak. However, enhanced signal in mineralized samples with type I collagen was detected not only in the predentine but also at the limit between intertubular dentine, within the elements of the enamel organ and subgingival stroma. This report offers advances in sectioning mineralized dental tissues and allows the application of immunofluorescence not only for on‐section protein detection but importantly for detecting cross‐linked fibrous collagens in both soft and mineralized tissue sections. Microsc. Res. Tech. 73:741–745, 2010. © 2009 Wiley‐Liss, Inc.     

The influence of elastic and ionization cross-section approximations on the result of Monte Carlo calculations

A Monte Carlo program has been developed on the basis of a Mott cross-section and knock-on model. The use of this program for the set of the film-substrate type systems shows that there is some difference between the calculated and experimental x-ray depth distribution. To clear up the reasons for the discrepancies, the Monte Carlo model is investigated by calculating the various physical quantities associated with the electron distribution in homogeneous bulk and film specimens and by comparing them with the known experimental data. The influence of the elastic and ionization cross-section approximation on the electron distribution in target is studied.     

Demonstration of lanthanum in liver cells by energy-dispersive X-ray spectroscopy, electron energy loss spectroscopy and high-resolution transmission electron microscopy

The appearance of lanthanum in liver cells as a result of the injection of lanthanum chloride into rats is investigated by advanced transmission electron microscopy techniques, including electron energy loss spectroscopy and high‐resolution transmission electron microscopy. It is demonstrated that the lysosomes contain large amounts of lanthanum appearing in a granular form with particle dimensions between 5 and 25 nm, whereas no lanthanum could be detected in other surrounding cellular components.     

Biological applications of electron probe microanalysis in france

The contribution of French science to the field of biological X-ray microanalysis is reviewed. The main analytical microscopy centers are listed, and their methods and main results are summarized.