Calcium distribution in high-pressure frozen bone cells by electron energy loss spectroscopy and electron spectroscopic imaging

 Subcellular localization of total calcium requires tissue processing that preserves the chemical composition of the samples and a highly sensitive microanalytical technique. In this study rat fetal bone samples were submitted to high-pressure freezing and freeze substitution. Ultrastructural preservation was good in the superficial sections: osteoblasts near the bone mineral had clearly defined plasma and nuclear membranes, dense mitochondria, and numerous ribosomes. Electron energy loss spectroscopy allowed high-resolution calcium-sensitive images to be obtained using ionization edge loss electrons. In biological samples, the Ca–L₂,₃ signal is superimposed on the carbon edge and artifacts may result from thickness and scattering effects. Therefore the relative thickness was established for each area analyzed (t/λ<0.5). Background was subtracted using the three-images method, allowing high resolution calcium-sensitive images of intramitochondrial granules and of intracellular compartments, and semiquantitative data from the granules to be obtained. Calcium maps were confirmed by spectra collected on defined areas of the images and the shape of the net Ca–L₂,₃ edges was compared to the characteristic Ca–L₂,₃ edge of bone crystals. These procedures will provide new information about total calcium localization in bone cells and the possibility of examining the distribution of other elements. Accepted: 22 July 1997     

Application of electron energy loss spectroscopy and electron spectroscopic imaging to aluminum determination in biological tissue

Electron energy loss spectroscopy (EELS) is a high spatial resolution electron microscopic technique with the potential to quantify elements at the subcellular level. The presence of each element is demonstrated by the electron energy loss edge at the energy characteristic of that element. The area of the edge may indicate the quantity of element present. Electron spectroscopic imaging (ESI) is a similar technique generating graphic images of elemental localization in the specimens. An ESI of an aluminum (Al)-loaded rabbit hippocampus showed Al only in pyramidal cell lysosomes, but no EELS edge could be obtained. To determine the sensitivity of EELS for Al and to be able to adjust the instrument to optimal operating conditions, standards containing 50–5000 ppm Al were produced. An Al-chloride:dicyclohexano-18-crown-6 (Al:crown) complex was synthesized. The purity of the complex was confirmed by nuclear magnetic resonance (NMR) spectroscopy and the percentage of Al in the complex was determined by electrothermal atomic absorption spectroscopy (ETAAS). The complex was introduced into a biological tissue embedding resin (Spurr medium) and appeared to be compatible with the resin at Al concentrations ≤500 ppm. EELS signals from the Al K edge could be obtained at a spatial resolution of 3.3 nm in a 30-nm thick section from 2.78×10^?21 g of Al, representing a sample concentration of 1% Al.     

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

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

Phosphorus distribution in perichromatin granules and surrounding nucleoplasm as visualized by electron spectroscopic imaging

Summary— The in situ distribution of phosphorus in perichromatin granules (PCGs), and in the surrounding nucleoplasm was investigated in rat liver cells by means of electron spectroscopic imaging of unstained preparations. A 2–3 nm fibril containing high concentration of phosphorus was found to be the main substructural feature of the PCGs revealed in the maps of phosphorus. This fibril is folded within the PCG with no apparent order. Fibrils of similar diameter and phosphorus content were also found in both the halo surrounding the PCG and dispersed in the nucleoplasm. Some of such fibrils are in continuity with those occurring within PCGs. Sometimes these fibrils are grouped forming a stalk connecting the PCG to chromatin. Some stalked PCGs are U-shaped or kidneyshaped, resembling Balbiani ring granules in the process of formation as observed in Chironomus salivary gland cell nuclei. The external fibrils are interpreted as perichromatin fibrils considered to be precursors of PCGs.     

Electron spectroscopic imaging: parallel energy filtering and microanalysis in the fixed-beam electron microscope

A new imaging modality in electron microscopy uses energy filtration to produce micrographs with elastically scattered electrons or with electrons that have lost a specific, often characteristic amount of energy in interacting with the specimen. No deleterious effects on microscope performance are encountered. Instead, microanalysis of specimens is made possible with a spatial resolution of 3 to 5 A and a sensitivity of detection of 2 X 10(-21) g corresponding to about 50 atoms of phosphorus. Elements detected range from hydrogen (Z = 1) to uranium (Z = 92). Examples of elemental mapping show membrane structure, DNA within nucleosomes, and RNA within ribosomal particles.     

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.     

Production of concentrated polyphenols by the root cap cells ofCorylus associated withTuber: ultrastructural study and element localization using electron energy loss spectroscopy and imaging

In mycorrhizae, root cap cell differentiation is a rapid event. Dark deposits abruptly invade the cytoplasm and the vacuoles. Finally the vacuoles fill with phenolic deposits and the cytoplasm is moribund. After degradation of the cell wall and cytoplasm, hyphae make contact with polyphenols and penetrate the polyphenols. The polyphenols are incorporated into the fungal mantle. Microanalysis by means of an electron-energy-loss spectrometer (EELS) and an electron imaging system (ESI) reveals the presence of calcium and nitrogen in the polyphenolic material just incorporated into the apical mantle. More distant polyphenols no longer contain calcium but nitrogen is still locally present.     

Tumor inhibition by metallocenes: ultrastructural localization of titanium and vanadium in treated tumor cells by electron energy loss spectroscopy

The intracellular distribution patterns of the metal atoms titanium and vanadium after in vivo as well as in vitro treatment of Ehrlich ascites tumor with the antitumor agents titanocene dichloride (TDC) or vanadocene dichloride (VDC) have been investigated by use of electron energy loss spectroscopy (EELS). The metals were found mainly accumulated in the nuclear heterochromatin and, to a minor extent, in the nucleolus and in the cytoplasmic ribosomes. In connection with other experimental results it is argued that this accumulation is indicative of the molecular interaction of the metal-containing species with the nucleic acids, especially with the DNA.     

Kinetochore structure: electron spectroscopic imaging of the kinetochore

The structure of the kinetochore in thin section has been studied in the Indian muntjac by an electron spectroscopic imaging technique. This procedures allows the analysis of the distribution of phosphorus within the layers of the kinetochore. The results indicate that this element is a major component of both the inner and outer plates whereas it is largely absent in the middle plate and fibrous corona. The majority of the phosphorus is localized to a 30-nm fiber(s) that is woven through the layers of the kinetochore. The presence of phosphorus within this fiber, along with its morphological and biochemical features, indicates that it contains DNA. The fiber(s) occupies a major portion of the inner and outer plate where it forms a series of rows. It is rarely observed in the middle layer except where it passes between the inner and outer layers. The absence of structure in the middle plate suggests that it may represent a space rather than a plate that in turn may be related to the function of this region. The distribution of phosphorus within the kinetochore is neither altered by treatment with colcemid nor by the presence of microtubules at the kinetochore. Analysis of conventional micrographs of the kinetochore together with structural information obtained by electron spectroscopic imaging suggests that most microtubules insert and terminate between the rows of kinetochore fibers in the outer plate. However, some microtubules continue through the middle layer and terminate at the lower plate. The insertion of microtubules at different levels of the kinetochore may reflect the existence of functionally distinct microtubule classes. Electron spectroscopic imaging indicates that the microtubules associated with the kinetochore are phosphorylated.     

Visualization of early intramembranous ossification by electron microscopic and spectroscopic imaging

We present electron microscopic and electron spectroscopic images of putative nucleation sites and early mineral deposits during intramembranous ossification of the murine perichondrial ring. Electron spectroscopic imaging (ESI) permits the quantitative determination and direct visualization of spatial distribution of atomic elements within specimens at high spatial resolution. In this study ESI was used to determine the elemental distributions of phosphorus, sulfur, and calcium. Nucleation and subsequent mineralization in the perichondrial ring occurred sequentially along the longitudinal axis. Proximal regions of the ring contained a matrix with only a few nucleation sites that are characterized in conventional electron micrographs as small loci of low-density material in which dense particles are located. Elemental maps of these sites that we obtained by ESI reveal a sulfur- containing matrix in which localized concentrations of phosphorus occur. With further maturation the loci became centers for the genesis of numerous dense rods or crystals. These mineral deposits contained increased concentrations of P, S, and Ca, compared with the surrounding matrix. The appearance of S at nucleation sites and its persistence in developing mineral deposits suggests that a sulfur-containing moiety may serve as a locus within the osteoid matrix to attain high local concentrations of Ca and P, which leads to the controlled local formation of calcium phosphates. Calcification of the perichondrial ring has been found to occur in the absence of matrix vesicles, which illustrates that these membrane-bounded organelles are not obligatory sites for nucleation in this matrix.     

Ultrastructural cytochemical localizations by back-scattered electron imaging of white blood cells

White blood cells have been studied in the back-scattered electron imaging (BEI) mode of scanning electron microscopy (SEM) with cytochemical methods for endogenous peroxidase, acid phosphatase, and a silver-staining method for nuclei. Peroxidase-positive granules were seen with good contrast and resolution in myeloid precursor cells and acid phosphatase activity was easily detected in macrophages and monoblasts. Silver staining permitted recognition of the shapes and location of the nuclei. In spite of the cytochemical procedures, cell surface structures were reasonably well-preserved in all methods, making direct correlation of BEI and secondary electron imaging (SEI) images an attractive feature in cell research with the scanning electron microscope.     

The three-dimensional organization of tight junctions in a capillary endothelium revealed by serial-section electron microscopy

Estimates of capillary permeability for hydrophilic solutes are generally interpreted in terms of Pappenheimer's pore theory. The intercellular clefts of the capillary endothelium are considered a likely structural equivalent to the postulated system of small hydrophilic pores. However, correlation of permeabilities and cleft structure requires more knowledge of the detailed structure of the tight junctions which appear to obliterate the clefts. In this study the organization of tight junctions in endothelium of rat heart capillaries has been investigated by serial-section electron microscopy. Cross-sectioned intercellular clefts were photographed in a series of 190 consecutive sections (average thickness approximately equal to 40 nm) and in a series of 16 consecutive sections (average thickness approximately equal to 12.5 nm). Seventy-one junctional segments, each extending over 5-32 consecutive sections, were reconstructed. The endothelial junctions were organized as irregular networks of lines of contact between neighboring cells. Six pathways circumventing the lines of contact were followed through the entire junctional region of the clefts providing a tortuous pathway connecting the luminal and abluminal aspects of the clefts. Moreover, the individual lines of contact were provided with discrete discontinuities, apparently 4 nm wide. The observations support the notion that the paracellular pathway in capillary endothelium is permeable not only to small solutes but also to certain macromolecules.     

Subcellular localization of copper in the root cells of Allium sativum by electron energy loss spectroscopy (EELS)

Ultrastructural investigation of the root cells of Allium sativum L. exposed to three different concentrations of Cu (1, 10 and 100 microM) for 9 days was carried out using transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS). The results presented here indicate that excess Cu induces ultrastructural changes such as strong vacuolization, condensed nuclear chromatin, decreased endoplasmic reticulum (ER) and ribosome and serious plasmolysis. EELS analysis indicated that electron dense granules containing Cu appeared in the cells after Cu treatment. The vacuoles of the root tip cells were the main Cu-accumulation site. Small amounts of copper were also localized to cytoplasmic vesicles or cell walls of cortical cells. The results of the present investigation have significant importance in further understanding the mechanisms of absorption, transportation and accumulation of heavy metals in plants grown in polluted soil.     

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

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

Differentiated microdomains on the luminal surface of capillary endothelium: distribution of lectin receptors

Lectins conjugated with either peroxidase or ferritin were used to detect specific monosaccharide residues on the luminal front of he fenestrated endothelium in the capillaries of murine pancreas and intestinal mucosa. The lectins tested recognize, if accessible, the following residues: alpha-N-acetylgalactosaminyl (soybean lectin), beta- D-galactosyl (peanut agglutinin [PA] and Ricinus communis agglutinin- 120 [RCA]), beta-N-acetylglucosaminyl and sialyl residues (wheat germ agglutinin [WGA]), alpha-L-fucosyl (lotus tetragonolobus lectin), and alpha-D-glucosyl and beta-D-mannosyl (concanavalin A [ConA]). Thi labeled lectins were introduced by perfusion in situ after thoroughly flushing with phosphate-buffered saline the microvascular beds under investigation. Specimens were fixed by perfusion, and subsequently processed for peroxidase detection and electron microscopy. Control experiments included perfusion with: (a) unlabeled lectin before lectin conjugate; (b) labeled lectin together with the cognate hapten sugar, and (c) horseradish peroxidase or ferritin alone. Binding sites were found to be relatively homogeneously distributed on the plasmalemma proper, except for Lotus tetragonolobus lectin and Con A, which frequently bound in patches. Plasmalemmal vesicles, transendothelial channels, and their associated diaphragms were particularly rich in residues recognized by RCA and PA (beta-D-galactosyl residues) and by WGA (beta-N-acetylglucosaminyl residues). Receptors for all lectins tested appeared to be absent or considerably less concentrated on fenestral diaphragms. The results reported here extend and complement previous findings on the existence of microdomains generated by the preferential distribution of chemically different anionic sites (Simionescu et al., 1981, J. Cell Biol., 9:605-613 and 614-621).