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Boron imaging at the subcellular level in tissues is of prime importance to understand boron neutron capture therapy (BNCT) effects and to suggest ways to improve the efficiency of the treatments. In this paper, we present boron imaging in cryofixed biological tissues by electron energy loss spectroscopy (EELS), using the spectrum‐imaging acquisition mode. We performed boron imaging in tissues of mice who received boron compounds. We analysed cryosections of tumours, livers, and kidneys of infused mice. Our results point out the relevance of EELS for boron imaging and, particularly, in order to detect small accumulation areas. The data also point out the limitations of the technique and its complementarity with other imaging techniques. We discuss and illustrate the possibility for misinterpretation of EELS or electron spectroscopic imaging (ESI) data. We particularly focus on the problem of phosphorus for boron imaging. This manuscript is dedicated to Dr. K. Zierold who died in 2004.

The distribution of specific atoms and molecules within living cells is of high interest in bio-medical research. Laser secondary neutral mass spectrometry (laser-SNMS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) detect atoms with high sensitivity and spatial resolution. The application of these methods to cultured cells requires special preparation techniques preserving morphological and chemical integrity of the living cells. The cells should, therefore, be grown on a conducting material preventing charging of the sample during ion bombardment. Silicon is currently used as the preferred support material for non-biological samples in mass spectrometry. This study investigates (1) the influence of silicon surfaces on cell growth and (2) the suitability of a sandwiched, rapid freezing method to analyse transmembrane ion gradients. Human melanoma cells were grown on silicon with polished or etched surfaces. Growth kinetics were studied using the Sulforhodamine-B assay. Number, shape, and morphology of the cells were assessed by epifluorescence microscopy of calcein AM- and DAPI-stained cells. Cells were subjected to rapid freezing, freeze-fracturing, and freeze-drying prior to analysis by TOF-SIMS and laser-SNMS. While cell numbers and morphology on the rough silicon wafers were impaired, morphology and growth kinetics of cells on polished silicon were identical to control cells on cell culture tested polystyrene. TOF-SIMS and laser-SNMS resulted in high-resolution elemental images and mass spectra. Measurement of the intracellular Na+ and K+ concentrations revealed a ratio as observed in living cells. In conclusion, culturing cells on polished silicon wafers followed by sandwiched, rapid freezing is an adequate preparation method to study intracellular ion distribution with mass spectrometry. Microsc. Res. Tech. 66:248–258, 2005. © 2005 Wiley-Liss, Inc.

Analytical electron microscopy is used to identify, localize and quantify chemical elements in biological samples with the aim to correlate the element distribution in cells and tissues with the particular functional state. Energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections is considered to be the most efficient technique with respect to spatial analytical resolution and detection limit. The potentialities and limitations of this technique are illustrated by results from 3 current research projects: Element compartmentation in hepatocytes, cytotoxicity of an organotin compound, and calcium movements during osmoregulation of epithelia. At present, the absolute detection limit is 170 atoms in a minimum analytical volume of 30 × 30 × 100 nm3. X-ray microanalysis of cryosections from biological samples is limited by ice crystal growth during cryofixation and by the poor X-ray collection efficiency in most electron microscopes. Therefore, the construction of an analytica...

The element compartmentation in cultured rat hepatocytes was studied by electron probe X-ray microanalysis of freeze-dried cryosections after exposure of the cells to the ionophores valinomycin, ionomycin or gramicidin A. The most striking effect of these ionophores is the decrease of the intracellular potassium/sodium ratio from values of approximately 10 under control conditions to values below 1 after application of the ionophores. Changes of sodium, potassium and chloride are similar in cytoplasm and nucleus. However, elemental changes are delayed or impeded in mitochondria with respect to the surrounding cytoplasm. The water portion of cytoplasm and mitochondria slightly increases. Besides that, each ionophore has specific effects on the intracellular ion distribution. As compared to gramicidin A and ionomycin, valinomycin does not change the intracellular chloride content. Ionomycin induces calcium accumulation in mitochondria. The cytotoxic effects of the studied ionophores on the intracellular element distribution are more complex than supposed from their ion selective properties in membranes.

The flow behavior of aqueous solutions of cetyltrimethylammonium bromide was investigated as a function of temperature (15−50 °C) in the presence of various amounts of three 9-anthrylalkanols: 9-anthrylmethanol; 9-(1-(1-hydroxy)ethyl)anthracene; 9-(1-(1-hydroxy-2,2,2-trifluoro)ethyl)anthracene. Changes from non-Newtonian flow to Newtonian flow were observed upon increasing the temperature or decreasing the content of the 9-anthrylalkanols. The temperatures (between 291 and 321 K) and concentration ratios, at which these changes were observed, are distinct for the three solubilizates. Evaluations of rheological and light scattering data are in accordance with the presence of very big globular micellar aggregates (diameters up to 35 nm) in the Newtonian and long rodlike aggregates (lengths up to 800 nm) in the non-Newtonian cases. Electron micrographs confirm this interpretation.

We compared Hall's peak to continuum ratio method with a peak ratio method in order to quantify light elements (C, N, and O) in organic specimens as a model for biological thin sections. X-ray spectra were recorded by an energy dispersive X-ray spectrometer equipped with an ultra thin window detector. Spectra were processed by means of a top-hat filter adapted to peak full-width half maximum. The peak intensities were measured by multiple least square fitting to reference spectra. For most elements of biological interest, theoretical and experimental k-factors were determined. Absorption correction was found to be important for quantitation of carbon, nitrogen, and oxygen. Boron was efficiently detected; however, quantitative analysis was not possible. We conclude from our experiments that the peak ratio method is more suitable for quantitation of elemental concentrations in biological thin sections than the peak to continuum method.

Compartmentation of heavy metals on or within mycorrhizal fungi may serve as a protective function for the roots of forest trees growing in soils containing elevated concentrations of metals such as Cd and Zn. In this paper we present the first quantitative measurements by X-ray microanalysis of heavy metals in high-pressure frozen and cryosectioned ectomycorrhizal fungal hyphae. We used this technique to analyse the main sites of Cd and Zn in fungal cells of mantle and Hartig net hyphae and in cortical root cells of symbiotic Picea abies - Hebeloma crustuliniforme associations to gain new insights into the mechanisms of detoxification of these two metals in Norway spruce seedlings. The mycorrhizal seedlings were exposed in growth pouches to either 1 mM Cd or 2 mM Zn for 5 weeks. The microanalytical data revealed that two distinct Cd- and Zn-binding mechanisms are involved in cellular compartmentation of Cd and Zn in the mycobiont. Whereas extracellular complexation of Cd occurred predominantly in the Hartig net hyphae, both extracellular complexation and cytosolic sequestration of Zn occurred in the fungal tissue. The vacuoles were presumed not to be a significant pool for Cd and Zn storage. Cadmium was almost exclusively localized in the cell walls of the Hartig net (up to 161 mmol kg -1 DW) compared with significantly lower concentrations in the cell walls of mantle hyphae (22 mmol kg -1 DW) and in the cell walls of cortical cells (15 mmol kg -1 DW). This suggests that the apoplast of the Hartig net is a primary accumulation site for Cd. Zinc accumulated mainly in the cell walls of the mantle hyphae (111 mmol kg -1 DW), the Hartig net hyphae (130 mmol kg -1 DW) and the cortical cells (152 mmol kg -1 DW). In addition, Zn occurred in high concentrations in the cytoplasm of the fungal mantle hyphae (up to 164 mmol kg -1 DW) suggesting that both the cell walls and the cytoplasm of fungal tissue are the main accumulation sites for Zn in P. abies resulting in decreased Zn transfer from the fungus to the root.

The aim of this study was to show the potential of Thlaspi caerulescens in the cleaning-up of a moderately Zn -contaminated soil and to elucidate tolerance mechanisms at the cellular and subcellular level for the detoxification of the accumulated metal within the leaf. Measured Zn concentrations in shoots were high and reached a maximum value of 83 mmol kg -1 dry mass, whereas total concentrations of Zn in the roots were lower (up to 13 mmol kg -1 ). In order to visualize and quantify Zn at the subcellular level in roots and leaves, ultrathin cryosections were analysed using energy-dispersive X-ray micro-analysis. Elemental maps of ultrathin cryosections showed that T. caerulescens mainly accumulated Zn in the vacuoles of epidermal leaf cells and Zn was almost absent from the vacuoles of the cells from the stomatal complex, thereby protecting the guard and subsidiary cells from high Zn concentrations. Observed patterns of Zn distribution between the functionally different epidermal cells were the same in both the upper and lower epidermis, and were independent of the total Zn content of the plant. Zinc stored in vacuoles was evenly distributed and no Zn-containing crystals or deposits were observed. From the elemental maps there was no indication that P, S or Cl was associated with the high Zn concentrations in the vacuoles. In addition, Zn also accumulated in high concentrations in both the cell walls of epidermal cells and in the mesophyll cells, indicating that apoplastic compartmentation is another important mechanism involved in zinc tolerance in the leaves of T. caerulescens.

The cytocompatibility of two particulate bioceramics, zirconia and alumina, was studied using human blood monocytes driven to differentiate into mature macrophages with granulocyte macrophage-colony-stimulating factor. Changes in individual cell elemental composition, particularly sodium and potassium content, were assessed by X-ray microanalysis of ultrathin freeze-dried sections. Phagocytosis and respiratory burst of macrophages exposed to biomaterial for 7 days were analyzed under flow cytometry using uptake of fluorescent latex beads and 2'7'-dichlorofluorescien diacetate oxidation, respectively. Zirconia and alumina particles were found to decrease the intracellular potassium/sodium ratio (an index of cell vitality) significantly (p 2 times reduced by zirconia and >5 times reduced by alumina). The present study clearly demonstrates that reduction of the phagocytic capacity of macrophages associated with altered oxidative metabolism caused by biomaterial particles is characterized by changes in intracellular elemental content. Thus, investigation of cellular homeostasis by electron probe microanalysis together with analysis of functional changes may improve estimation of biomaterial cytocompatibility.

To analyze the elemental composition and topology of the extracellular compartments of the compound eye, the eyes of blowflies Calliphora vicina were rapidly frozen and ultrathin cryosections were freeze dried. Three zones of an ommatidium, peripheral cytosol of visual cells, rhabdomeres, and ommatidial cavities were analyzed by X-ray microprobe analysis. The ommatidial cavity was found to contain sodium and potassium in proportion similar to that in the blowfly hemolymph. Potassium-to-sodium ratio in a cytosol was typical for a cytosol. The rhabdomeres displayed an electrolyte content intermediate between the above compartments. Three topologically connected extracellular compartments were characterized by the experiments with tracers, monastral blue and lanthanum: (1) common intercellular space of ommatidia including peripheral clefts between the visual cells, both tracers entered this compartment; (2) the ommatidial cavity, which is not accessible for monastral blue, however, as revealed by our X-ray microanalysis, it was reachable for lanthanum; (3) rhabdomeric loops, which were accessible for lanthanum entering either via the cavity or from the common intercellular clefts. The above characteristics of the ionic content and topology of ommatidial compartments might suggest higher sodium and lower potassium content in the microvilli as compared with the cytosol. The rhabdomeric and “cavital” plasma membranes are assumed to be permeable for these ions so that a voltage of only 25–30 mV, negative inside, is probably formed across them, much lower than the known resting potential −60 mV across the peripheral plasma membrane of a visual cell.

The biogenetic formation of mineral crystals, one aspect of biomineralization, is a multistep process of apatite formation throughout the growth of dentin tissue. An important step is the transformation of the non-mineralized predentin matrix to mineralizing dentin matrix and its biological control. In this study, the high capacity of elemental mapping is combined with single x-ray point measurements to elucidate whether special elements are involved in initiation or regulation of mineral nucleation. Directly at the mineralization front, micro-areas with a strong co-enrichment of phosphorus ( e.g., as phosphate) and potassium are found. During the beginning of the calcium enrichment and the subsequent apatite mineral formation in the characteristic micro-areas, the content of potassium decreases significantly. These findings indicate that potassium is involved in the process of dentin mineralization.

We present, for the first time, elemental mapping of ultra-thin cryosections from high-pressure frozen ectomycorrhizal roots of Picea abies–Hebeloma crustuliniforme. The maps provide interpretable information on the relationship between elements and the structure of inhomogeneous objects. Cryoultramicrotomy together with energy dispersive X-ray microanalysis (EDX) offers the potential to study the subcellular localization of specific ions and ecologically important tracers (Cs and Sr) in ectomycorrhizal roots under conditions resembling the natural slate as closely as possible. Structural changes of the ectomycorrhizal roots, in particular the absence of a Hartig net at high NH4+ levels in the nutrient solution, were accompanied by elemental modification of Ca in cortical cell walls, where markedly higher concentrations of Ca were found. Cs and Sr applied to the nutrient solution were localized in root and fungal cells of the Hartig net. Cs accumulated mainly in the vacuoles of the Hartig net hyphae and its distribution was very similar to the distribution of K. In contrast to Cs, Sr was found to occur mainly in electron-opaque and P-rich granules. From this study, (here is no indication that Ca is the only ion accompanying P in the P-rich granules. Several elements including Ca, K, Cl, S, Cs and Sr, with highest concentrations for S, can occur together with P in these granules. The occurrence of the P-rich electron-opaque deposits in fungal cells might be the first evidence of polyphosphate granules in the native state, since our specimen preparation technique did not include chemical fixation.

The distribution of elements in isolated and cultured rat hepatocytes was measured by energy dispersive electron probe X-ray microanalysis of freeze-dried ultrathin cryosections. The intracellular compartmentation of electrolyte ions, in particular the content of sodium, chloride, and potassium, was found to depend on culture conditions and on the amount of cadmium chloride added to the culture medium. In cells exposed to 1-10 microM cadmium without carbon dioxide supply, the potassium/sodium ratio decreased from control values of about 10 to values below 1 within 30 min. Changes of potassium and sodium content were followed by an increase in the intracellular chloride content. In cells exposed to 1-10 microM cadmium with carbon dioxide supply, changes of the electrolyte composition were delayed to 1-2 days. An increase of intracellular chloride preceded the inversion of the intracellular potassium/sodium ratio. High cadmium doses induced a cytoplasmic calcium increase and finally disintegration and decay of cell structure. Almost normal potassium and sodium contents were found in cells exposed to 10 microM cadmium in the presence of 100 microM zinc with carbon dioxide for 1 day. Changes in the intracellular electrolyte composition by adverse or toxic conditions were detected before any structural damage became visible. Thus, energy dispersive electron probe X-ray microanalysis of cryosections proved to be a sensitive probe of cell viability and cytotoxicity.

Elemental mapping of freeze-dried cryosections from biological cells was done using the energy window mode and the quantitative mode. Quantitative mapping requires long measuring times and high electron exposure resulting in inhomogeneous shrinkage and distortion of the cryosection. Collection of X-ray spectra for quantitative analysis of intracellular structures and compartments combined with energy window mapping was found to be sufficiently fast and reliable to reveal inhomogeneities of the element distribution in cells. However, this method is suitable only for mapping elements without overlapping peaks in the X-ray spectrum.

Summary A cryopunching device is described which allows cryofixation of tissue specimens by quick contact with a precooled copper surface during excision. The advantage of the cryopuncher for analytical electron microscopy of cells and tissues in defined functional states is illustrated by electron probe X-ray microanalysis of freeze-dried cryosections from rat liver and dogfish kidney. In comparison with results obtained from specimens plunged into liquid propane, cryopunching in situ results in similar preservation of morphology and remarkably improved intracellular K/Na ratio.

The routes calcium might take across the mantle to the shell have been investigated with various electron-microscopical techniques in the freshwater snail Biomphalaria glabrata (Planorbidae, Basommatophora). In chemically-fixed tissue, calcium was precipitated with a tannic acid-antimonate technique in predominantly the intercellular spaces of the outer mantle epithelium and the interstitium below it. Some vacuoles of the outer mantle epithelium and one type of mucus cell in the inner mantle epithelium also contained precipitate. The presence of calcium in the precipitates was proved by electron energy loss spectroscopy combined with electron spectroscopic imaging. Incubation with lead acetate and uranyl acetate revealed binding-sites for calcium in the intercellular spaces of the epithelia interstitium and the mucus cells of the inner mantle epithelium. Precipitates were also seen after all incubations in the calcium spherites of the connective tissue. The concentrations of calcium and other elements were analysed in freeze-dried ultrathin sections of cryofixed mantle tissue by means of energy-dispersive X-ray microanalysis. Only in mitochondria of the musculature could high amounts of calcium and phosphorous be detected.

In Drosophila larval Malpighian tubules, type-I concretions, normally accumulating calcium, magnesium and phosphorus are able to concentrate strontium and barium added to the diet. The concretions are not able to concentrate other metals like aluminium, iron, zinc and cadmium. The values for calcium, magnesium and phosphorus increase after feeding on a diet enriched in calcium, magnesium or phosphates. Such an experimental increase in one component in the diet leads to a decrease in other elements in the concretions. After simultaneous feeding on a diet enriched with equal quantities of calcium and strontium carbonate, strontium but not calcium is accumulated; the calcium concentration is even lower than in type-I concretions in animals raised on normal diet. Larvae of Drosophila are able to resorb cations of heavy soluble alkali earth salts like carbonates, sulphates and phosphates. All metals accumulated in type-I concretions are eliminated after pupation. In imaginal Malpighian tubules, only a few small concretions occur. Calcium-rich feeding does not enhance the normal low calcium content in type-II concretions.

In the Malpighian tubules of Drosophila two types of luminal concretions are formed. Type-I concretions mainly accumulate high quantities of calcium and magnesium in a matrix of proteoglycans; type-II concretions accumulate potassium. Higher quantities of organic matrix are found in type-II concretions than in type I concretions. The latter are stored in the distal segment of the anterior Malpighian tubules while type-II concretions are formed between apical microvilli in the transitional and the middle segment of anterior tubules and in the distal and the middle segment of posterior tubules. In the tubules of the pupa and in the first meconium to be produced, the magnesium and calcium contents of excreted type-I concretions are similar. The outer envelope of the puparium exhibits only traces of calcium in contrast to the cuticle of the larva. Both types of concretions are products of excretion: they eliminate calcium and magnesium or potassium, respectively. During winter time and under normal photoperiods both types of concretions can join together to form large “biocrystals” inhibiting the flow of urine. Concretion formation is discussed as an economical method to excrete high amounts of cations in a condensed form with the loss of little water.

SUMMARY Electron probe microanalysis data on the intracellular content and distribution of electrolyte ions depends critically on the functional state of the cells at the moment of cryofixation. Whereas tissue specimens often require special in-situ freezing techniques, isolated and cultured cells can be frozen within their environmental medium under physiologically controlled conditions. Thus, they represent a feasible system to study functional ion-related intracellular parameters such as the K/Na ratio. Specifically modified freezing devices allow the study of ion shifts related to dynamic processes in cells, for example, locomotion and exocytosis. The time resolution achieved by time-controlled cryofixation is approximately 1 ms.

SUMMARY Inner surfaces and fracture faces of rabbit kidney tissue were investigated with high-resolution scanning electron microscopy using two different cryopreparation techniques: (i) for the observation of fracture faces, cryofixed tissue was fractured and coated in a cryopreparation chamber dedicated to SEM, vacuum transferred onto a cold stage and observed in the frozen-hydrated state; (ii) for the observation of inner surfaces of the nephron, water was removed after freezing and fracturing by freeze substitution and critical-point drying of the tissue. By both methods, macromolecular structures such as intramembranous particles on fracture faces and particles on inner surfaces were imaged. The latter method was used to investigate in more detail surface structures of cells in the cortical collecting duct. These studies revealed a heterogeneity of intercalated cells not described thus far.

Oral amalgam tattoos (AT) are distinct pigmentations of the oral mucosa resulting from accidental incorporation of dental amalgam in the oral soft tissues. Dental amalgams and in particular mercury, one of the constituents of dental amalgams, have for long been considered toxic. Oral ATs are easily accessable to study soft tissue reaction to amalgam and its degradation products. In this study, 17 oral ATs were examined by transmission electron microscopy and energy dispersive X-ray microanalysis. Ultrastructurally, in the ATs, three kinds of electron-dense particles were observed. The largest particles ranged in size from 0.5 up to several 100 μm. Smaller electron-dense inclusions (0.5–0.1 μm) were seen extracellularly associated with meshworks of elastic fibers and collagen bundles. The third and smallest type of particles (5–30 nm in diameter) was found with basement membranes of small vessels and pericytes and particularly decorating collagen bundles. Element analysis regularly revealed the presence of silver, sulphur, copper and lead in the AT decay products. Mercury was found in only one instance. Tissue reactions due to ATs seem to be minimal. No acute inflammatory changes were seen. Larger inclusions occasionally were surrounded by macrophages and multinucleated cells. TEM and element analysis may in specific cases be helpful in the differential diagnosis of pigmented lesions of the oral mucosa.

The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na{sup +} absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na{sup +}, Mg{sup 2+}, P, S and Cl{sup -}) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR{sub inh}-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules maymore » contribute to the dehydration and increased viscosity of the ASL in CF.« less

The intracellular distribution of the elements carbon, nitrogen, and oxygen was measured in cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of 100-nm-thick freeze-dried cryosections. Electron irradiation with a dose up to 106e/nm2caused no or merely negligible mass loss in mitochondria and in cytoplasm. Cell nuclei lost carbon, nitrogen, and—to a clearly higher extent—oxygen with increasing electron irradiation. Therefore, electron doses less than 3 × 105e/nm2were used to measure the subcellular compartmentation of carbon, nitrogen, and oxygen in cytoplasm, mitochondria, and nuclei of the cells. The subcellular distribution of carbon, nitrogen, and oxygen reflects the intracellular compartmentation of various biomolecules. Cells exposed to inorganic mercury before cryofixation showed an increase of oxygen in nuclei and cytoplasm. Concomitantly the phosphorus/nitrogen ratio decreased in mitochondria. The data suggest mercury-induced production of ribonucleic acid (RNA) and decrease of adenosine triphosphate (ATP). Although biomolecules cannot be identified by X-ray microanalysis, measurements of the whole element spectrum including the light elements carbon, nitrogen, and oxygen can be useful to study specific biomolecular activity in cellular compartments depending on the functional state of the cell.

There are two types of concretions in Drosophila Malpighian tubules: Type-I concretions originate in the distal segments of the anterior tubules, type-II concretions in the adjacent transitional segment between the apical microvilli. Type-I concretions are formed with the aid of carbonic anhydrase within intracellular vesicles, which migrate to the apical cell membrane where they are discharged into the lumen by exocytosis. The carbonic anhydrase inhibitors acetazolamide or hydrochlorothiazide prevent the formation of concretions by interruption of bicarbonate supply. In addition, the formation of concretions can be reduced by feeding with sodium cellulose phosphate.

Magnesium besides calcium is the most important excretion product. In the anterior Malpighian tubules of Drosophila, excretion of magnesium takes place via the hindgut by proteoglycan containing concretions. This study reports on magnesium transport through the basal plasma membrane of the principal cells of the proximal segment of the anterior Malpighian tubules. Measurements by electron probe X-ray microanalysis indicate the existence of two antiporters which transfer magnesium in still unknown stoichiometry from the hemolymph space into the cell: Mg/H and Mg/Na.

Electron probe X-ray microanalysis can be used to determine the elemental composition of cells and tissues in defined functional states. However, reliable quantitative data can be expected only after appropriate cryopreparation techniques. The most versatile preparation and analysis technique can be outlined by four succeeding steps: 1. Cryofixation, 2. Cryoultramicrotomy, 3. Cryotransfer including freeze-drying, 4. Energy dispersive X-ray microanalysis.

Dictyostelium discoideum pseudoplasmodia exhibit a gradient of the cytosolic free Ca2]-concentration ([Ca2]]i) along their anterior posterior axis involved in cell-type specific differentiation. [Ca2]]i is high in prestalk and low in prespore cells.We determined the content and localization of calcium and other elements in cryosectioned cells of pseudoplasmodia and fruiting bodies by X-ray microanalysis. Granular stores rich in Ca, Mg and P were identified. Average Ca was higher in prespore than prestalk granules (225 vs 111 mmol/kg dry weight). Total Ca stored in granules was also higher in prespore than prestalk cells. The amount of P and S in granules differed between the two cell types indicating different store composition. In spores mean granular Ca was 120 mmol/kg dry weight. Stalk cells had smaller granules with 360 mmol Ca/kg dry weight. Complementary to microanalysis, vesicular Ca2]-fluxes were studied in fractionated cell homogenates. The rate of Ca2]-uptake was higher in pellet fractions of prespore than prestalk amoebae (4.7 vs 3.4 nmol/minemg). Ca2]-release was greater in supernatant fractions from prestalk than presporecells (16.5 vs 7.7 nmol/108 cells). In summary, prestalk and prespore cells possess qualitatively different, high-capacity stores containing distinct amounts of Ca and probably being involved in regulation of the anterior posterior [Ca2]]i-gradient.

Cytotoxicity of the heavy metals gold, mercury, thallium and lead was studied by measuring the intracellular element distribution of cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections in a scanning transmission electron microscope. Exposure of the cells to aqueous solutions containing heavy metal ions in concentrations reaching a critical concentration caused increase of intracellular sodium and chloride content accompanied or followed by decrease of intracellular potassium content. Thus, the intracellular potassium/sodium ratio drastically decreased from control values of approximately 10 to values below 1 before changes of cell morphology became visible. In experiments with gold or mercury the decrease of the potassium/sodium ratio was preceded by transient cytoplasmic increase of sulfur and phosphorus. Heavy metal concentrations exceeding the critical concentration also caused an increase of cytoplasmic calcium concentration and finally decay of the cell structure. Cytotoxicity of heavy metals was found to increase in the order Pb, Au, Tl, Hg. Cytotoxic effects by Au, Tl or Hg in moderate concentrations were reduced by simultaneous addition of Zn or Pb to the culture medium. The results obtained prove electron probe X-ray microanalysis of cryosections as a sensitive probe of cell viability.

The invasion of host cells by the obligate intracellular protozoan parasite Toxoplasma gondii is calcium dependent. We have identified two calcium storage areas in tachyzoites, the endoplasmic reticulum and vesicles that contain high concentrations of calcium as amorphous calcium phosphate precipitates. Our data indicate that these vesicles slowly lose their calcium during the intracellular development of the tachyzoite as their nucleus phosphorus content increases. We found fluctuations in the sulfur content of the tachyzoite during invasion following the exocytosis of protein from the secretory organelles, with a loss of sodium and chlorine, and the uptake of potassium from the host cell cytoplasm. We demonstrated that penetration of the tachyzoite into the host cell was accompanied by increases in the concentrations of phosphorus and sulfur in the host cell nucleus, probably due to increased transcription. The cytosol sodium concentrations decreased, while the potassium content increased. Thus, the subcellular element distribution of tachyzoites and host cells changes during invasion and intracellular growth of the parasites. In addition, our results indicate that tachyzoite calcium might be involved in the egress of the parasite from the host cell.

H2O2 mimicked the action of periportal pO2 in the modulation by O2 of the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase (PCK) gene and the insulin-dependent activation of the glucokinase (GK) gene. H2O2 can be converted in the presence of Fe2+ in a Fenton reaction into hydroxyl anions and hydroxyl radicals (•OH). The hydroxyl radicals are highly reactive and might interfere locally with transcription factors. It was the aim of the present study to investigate the role of and to localize such a Fenton reaction. Hepatocytes cultured for 24 h were treated under conditions mimicking periportal or perivenous pO2 with glucagon or insulin plus the iron chelator desferrioxamine (DSF) or the hydroxyl radical scavenger dimethylthiourea (DMTU) to inhibit the Fenton reaction. PCK mRNA was induced by glucagon maximally under conditions of periportal pO2 and half-maximally under venous pO2. GK mRNA was induced by insulin with reciprocal modulation by O2. DSF and DMTU reduced the induction of PCK mRNA to about half-maximal and increased the induction of GK mRNA to maximal under both O2 tensions. Hydroxyl radical formation was maximal under arterial pO2. Perivenous pO2, DSF and DMTU each decreased the formation of •OH to about 70% of control. The Fenton reaction could be localized in a perinuclear space by confocal laser microscopy and three-dimensional reconstruction techniques. In the same compartment, iron could be detected by electron-probe X-ray microanalysis. Thus a local Fenton reaction is involved in the O2 signalling, which modulated the glucagon- and insulin-dependent PCK gene and GK gene activation.

Cobalt and nickel stimulate, as does hypoxia, the production of erythropoietin (EPO) in HepG2 cells. Under hypoxic conditions, a decrease in the level of intracellular reactive oxygen species (ROS) is thought to stimulate EPO expression. Cobalt and nickel may interact with the putative oxygen sensor by changing the redox state of the central iron atom of heme proteins, similar to the effects of hypoxia. It was investigated, therefore, whether cobalt and nickel interact with hemeproteins or ROS scavenging systems in the control of intracellular ROS level. Cobalt chloride (100 microM, 24 h) oxidized non respiratory as well respiratory hemeproteins and increased the oxygen consumption. In contrast, nickel chloride (300 microM, 24 h) primarily reduced respiratory hemeproteins and decreased the oxygen consumption. In HepG2 cells treated with CoCl2, iron and cobalt were localized in cytosolic granules close to the cell nucleus and in mitochondria at concentrations up to 12 mM or 41 mM, respectively. Intracellular nickel was not measurable. Three-dimensional reconstruction of confocal laser microscopy images revealed hot spots of hydroxyl radical generation by a Fenton reaction at the sites of cytosolic iron accumulation. The .OH levels decreased in cobalt-treated (to 81%) as well as in nickel-treated (to 67%) HepG2 cells, accompanied by an increase of EPO expression to 167% and 150%, respectively. Our results underline the importance of .OH formed by a Fenton reaction for triggerimg EPO production. Identification of the primary hemeprotein being the oxygen sensor was not possible due to the antagonistic effects of cobalt and nickel on the redox state of detectable hemeproteins.

Recent biophysical investigations of vertebrate olfactory signal transduction have revealed that Ca(2+)-gated Cl(−) channels are activated during odorant detection in the chemosensory membrane of olfactory sensory neurons (OSNs). To understand the role of these channels in chemoelectrical signal transduction, it is necessary to know the Cl(−)-equilibrium potential that determines direction and size of Cl(−) fluxes across the chemosensory membrane. We have measured Cl(−), Na(+), and K(+) concentrations in ultrathin cryosections of rat olfactory epithelium, as well as relative element contents in isolated microsamples of olfactory mucus, using energy-dispersive x-ray microanalysis. Determination of the Cl(−) concentrations in dendritic knobs and olfactory mucus yielded an estimate of the Cl(−)-equilibrium potential E(Cl)in situ. With Cl(−) concentrations of 69 mm in dendritic knobs and 55 mm in olfactory mucus, we obtained an E(Cl) value of +6 ± 12 mV. This indicates that Ca(2+)-gated Cl(−)channels in olfactory cilia conduct inward currents in vivo carried by Cl(−) efflux into the mucus. Our results show that rat OSNs are among the few known types of neurons that maintain an elevated level of cytosolic Cl(−). In these cells, activation of Cl(−) channels leads to depolarization of the membrane voltage and can induce electrical excitation. The depolarizing Cl(−) current in mammalian OSNs appears to contribute a major fraction to the receptor current and may sustain olfactory function in sweet-water animals.

Electron probe X-ray microanalytical studies on the role of carbonic anhydrase in electrolyte transport in the cells of Drosophila Malpighian tubules indicate that carbonic anhydrase delivers protons and bicarbonate ions to ion transport systems in the cell membrane. After injection and after feeding acetazolamide or hydrochlorothiazide, known inhibitors of carbonic anhydrase, the contents of potassium, magnesium and chloride in the apical cytoplasm and in the cytoplasm close to the basal plasma membrane decreased. We explain our measurements by the hypothesis of a basal Mg-H-antiport system in parallel with Cl-HCO3-antiport, inhibitable by DIDS. Zinc is supposed to enters cells and intracellular Zn storage vacuoles by a negatively charged Zn-anion-complex in exchange for HCO3- ions. This antiport is inhibitable by SITS. The content of the Zn storage vacuoles is acid, as shown by red fluorescence after incubation of Malpighian tubules with acridine orange. Red fluorescence is absent after preincubation in a medium containing an inhibitor of carbonic anhydrase. Carbonic anhydrase was demonstrated cytochemically in the Golgi-ER complex, Golgi vesicles and intercellular space. We suppose that carbonic anhydrase is synthesized and stored in the Golgi-ER-complex from where it is released into the tubule lumen.

Electron probe microanalysis was applied to study quantitatively and semi quantitatively the elemental concentrations and distributions that occur in predentine during the dentine mineralization of rat incisor. Apex regions of the continuously growing incisors were rapidly dissected and cryofixed in liquid nitrogen-cooled propane. Ultrathin cryosections were prepared from the dentine tissue. On the average in the extracellular predentine element concentrations of calcium and phosphorus were about 0.5% (w/w) and 0.5-1% (w/w), respectively; so the calcium content in the extracellular predentine is higher while the phosphorus content is much lower than in the odontoblast area. Due to the high content of glycosaminoglycans in the extracellular matrix the concentration of sulfur in the predentine was more than 1% (w/w); the potassium content was found in the range of 0.6-0.8% (w/w) which is quite high for an extracellular area and the concentrations of sodium and chlorine were higher than 2% (w/w). Elemental mapping analysis was carried out to demonstrate the distribution of some important elements at the predentine/dentine border during mineralization.

Starving Dictyostelium discoideum amoebae aggregate within a few hours by chemotaxis towards the attractant cAMP to form a multicellular organism. The differentiating cells possess rapid and efficient calcium buffering and sequestration systems which enable them to restrict changes in the cytosolic free calcium concentration temporally and spatially during their chemotactic reaction and allow the continuous accumulation of Ca 2+ during development. In order to identify and to characterize calcium storage compartments, we analyzed the element content of amoebae at three consecutive stages of differentiation. Determination of the element distribution was done using energy-dispersive X-ray microanalysis of freeze-dried cryosections of rapid-frozen cells. Amoebae were frozen in the vegetative and aggregation-competent state and after formation of aggregates. Aggregation-competent as well as aggregated cells contained mass dense granules with large amounts of calcium together with phosphorous and either potassium or magnesium: in aggregation-competent cells calcium was colocalized with potassium, whereas in aggregated cells the mass dense granules contained calcium and magnesium. Although mass dense granules were also present in undifferentiated, vegetative cells, they contained only low amounts of phosphorous and potassium together with little Ca and Mg. We conclude that during their differentiation D. discoideum cells use an intracellular storage compartment to sequester Ca and other cations constantly throughout development.

The hypothesis that a large, possibly toxic, increase in cellular calcium accompanies photoreceptor cell degeneration in several differentDrosophilamutants was tested. The calcium content of wild type and mutant photoreceptors ofDrosophilawas measured using rapid freezing of the eyes and energy-dispersive x-ray analysis (e.d.x.) of cryosections and semithin sections of cryosubstituted material. Light- and dark-raised mutants of the following strains were studied: retinal degeneration B (rdgB);retinal degeneration C (rdgC);neither inactivation nor afterpotential C (ninaC), and no receptor potential A (norpA).These are light-dependent retinal degeneration mutants in which the affected gene products had been previously shown as myosin-kinase (ninaC), calcium-dependent phosphoprotein phosphatase (rdgC), phosphoinositide transfer protein (rdgB), and phospholipase C (norpA).In light-raised mutants, ommatidia of variable degrees of degeneration were observed. Mass-dense globular bodies of 200–500 nm diameter in relatively large quantities were found in the degenerating photoreceptor of all the mutants tested. These subcellular globules were found to have a very high calcium content, which was not found in wild type or in nondegenerating photoreceptors of the mutants. Nondegenerating photoreceptors were found not only in dark-raised mutants, but in smaller quantities also in light-raised mutants. Usually these globular structures contained high levels of phosphorus, indicating that at least part of the calcium in the mutant photoreceptors is precipitated as calcium phosphate. The results indicate that a large increase in cellular calcium accompanies light-induced photoreceptor degeneration in degeneratingDrosophilamutants even when induced by very different mutations, suggesting that the calcium accumulation is a secondary rather than a primary effect in the degeneration process.

To identify proteins involved in the hepatocellular uptake of loop diuretics, [3H]bumetanide was photoactivated by light flash in the presence of either intact isolated rat hepatocytes, rat liver basolateral plasma membranes or integral membrane proteins extracted from the basolateral plasma membranes. Proteins of 52-54, 48, 33, 27, 25 and 23 kDa in sodium dodecyl sulfate (SDS) gel electrophoresis were radiolabeled on intact hepatocytes. On liver basolateral plasma membranes a 50-52 kDa protein was the most intensely labeled protein. After separation into integral and associated membrane proteins by extraction with Triton X-114, radioactive labeling was only found in integral membrane proteins with a molecular weight of 50-52 kDa. Photoactivated bumetanide irreversibly inhibited the hepatocellular uptake of cholate, taurocholate but not of serine. Binding proteins for photoactivated bumetanide were absent on AS 30-D ascites hepatoma cells. Labeling of all proteins was sodium dependent in intact hepatocytes but was sodium independent in plasma membranes. Labeling was prevented by non-labeled bumetanide and by the loop diuretics piretanide and furosemide. Labeling protection was further achieved with organic anions such as bromosulfophthalein, rifampicin, probenecid and by the bile acids taurocholate, deoxycholate and dehydrocholate. The radiolabeled proteins did not belong to the bumetanide-sensitive NaCl/KCl co-transport system which apparently does not occur in intact isolated rat hepatocytes.

Elements in biological cells can be localized by EPXMA. The most attractive approach of this method with respect to detection limit and spatial resolution is scanning transmission electron microscopy combined with energy dispersive x ray microanalysis of ultrathin (approximately 100 nm thick) freeze-dried cryosections. The detection limit, here determined by scanning an electron beam of 1.3 nA for 2 min over freeze-dried cryosections from frozen standard solutions was found to be about 10 mMol/kg dry weight for all elements with the atomic number Z higher than 12. Due to the Be window in the x ray detector the detection limit was 20 mMol/kg dry weight for Mg (Z = 12) and 30 mMol/kg dry weight for Na (Z = 11). The lateral analytical resolution was less than 50 nm, limited by section thickness. In most cells 10 mMol/kg dry weight correspond to an absolute detection limit of .500 atoms within a volume of 100×50×50 nm3.

The morphology of ultrathin (ca. 100 nm thick) sections of glycerol-gelatine and yeast cell suspensions is studied in the frozen-hydrated and freeze-dried state by scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM). It is found that the quality of cryosections increases with decreasing ice crystal size in the specimen from which the sections are cut. However, linear deformations caused by sectioning cannot be avoided by varying the cutting temperature between 115 and 193 K or the cutting speed between 0.5 and 70 mm/s. A model idea describing the formation of periodic deformation lines in cryosections is proposed. Frozen-hydrated and freeze-dried cryosections are compared with respect to contrast, radiation damage and transfer requirements.

SUMMARY X-ray microanalysis in the scanning electron microscope (SEM) is used to obtain information about the distribution and mobility of electrolyte ions in the skeletal muscle tissue. In order to localize the diffusible ions in their physiological state, so far as possible, any chemical fixative is avoided. A special ‘cooling chain’ preparation method is applied which enables studies on cryofractured bulk specimens at low temperatures. Quantitation of the obtained X-ray spectra is done by the use of glycerol-treated tissue specimens which are incubated with electrolyte solutions of defined ion concentration.

Isolated rat hepatocytes maintained in primary culture on gas permeable membrane for 20 h form monolayers and establish at their cell borders a network of canaliculi (approximate diameter 3.5 micron). In the presence of the known choleretic bile acid dehydrocholate, dilation of canaliculi occurs. When nonfluorescent carboxyfluorescein diacetate ester is added to the culture medium, fluorescent carboxyfluorescein appears in the intracanalicular space. In the dilated state, fluid containing the fluorescent compound could be collected from the canaliculi by puncture with a micropipette. The intracanalicular space shows a negative electrical potential difference of 31 mV in reference to the bath solution and is 13.5 mV more positive with reference to recordings from the cytosol of cultured rat hepatocytes. Cultured rat hepatocytes grown on gas permeable membrane are energetically stable over 3 d. On Day 4, ATP levels increase markedly, whereas Na+-K+-ATPase activity declines. Ionic composition of hepatocytes, as measured by electronprobe element analysis on cryosection samples, does not change markedly during monolayer formation. With formation of bile canaliculi, the activity of alkaline phosphatase rapidly increases within 24 h and is stable for the next 3 d. Within that time the activity of gamma-glutamyltranspeptidase, however, increases steadily, reaching a 1.6-fold higher activity than freshly isolated hepatocytes. Bile acids appear in the culture supernatant after 1 d. When unconjugated [14C]cholic acid is added to the cultures the supernatant contains also [14C]tauro- and [14C]glycocholic acid, indicating the preservation of conjugation capacity in these cultures. Total bile acid concentrations in the supernatant increase from 5 to 26 microM on Day 4. The cultures do not secrete alpha-fetoprotein. Monolayer cultures of hepatocytes in the presence of choleretic bile acids seem to be a suitable model system to collect and to analyze the composition of primary bile. In conjunction with the electrical parameters, it is possible to describe directly properties of bile secretion at the canalicular pole of the intact hepatocyte.

Yolk-platelet crystals in the teleosts Pelvicachromis pulcher and Noemacheilus barbatulus and the amphibians Xenopus laevis, Rana temporaria, R. esculenta, and Triturus sp. have been studied by electron diffraction and imaging using a standardized processing (glutaraldehyde-osmium tetroxide fixation, glutaraldehyde-urea embedding, thin-section staining), by X-ray microanalysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their constituents. The crystal lattice is orthorhombic having--following standardized processing--in three amphibians a = 9.0 nm, b = 17.6 nm, c = 19.2 nm, and in the two teleosts a = 8.9 nm, b = 17.6 nm, c = 20.0 nm (averages). These values are very close to X-ray data from wet crystals (Xenopus laevis). Crystal images in the three axial projections point to the presence of space group P212121 (or an approximation of it since the lipovitellin dimers cannot be fully equivalent in some cases), to differences between the phosvitins of the two teleosts, and to a highly unusual stain exclusion from large crystal constituents interpreted as representing lipovitellin dimers. Microanalysis in ultrathin cryosections and other preparations revealed K and Cl to be the prominent ions in the crystals (and in the superficial layer of the platelet). Gel electrophoresis (including data of cyclostomes) showed considerable molecular variations despite a closely similar crystal architecture, emphasizing a physiological significance of the architecture, which may have remained conserved for nearly 400 million years according to paleontologic views.