Electron spectroscopic imaging of encapsidated DNA in vaccinia virus.

We have used electron spectroscopic imaging to locate the phosphorus in vaccinia DNA in situ in unstained, ultrathin sections of virions. The phosphorus of the DNA backbone appeared to form a halo on the core periphery surrounding a phosphorus-impoverished central element. These results constrain models for how DNA could be packaged into mature vaccinia particles.

Vectorial sequence of mineralization in the turkey leg tendon determined by electron microscopic imaging.

Turkey leg tendons were used as a model tissue to study the spatial and temporal relationships of mineral deposition between matrix vesicles and collagen fibrils by various electron microscopic techniques--bright field, selected-area dark field (SADF), and electron spectroscopic imaging (ESI). These latter imaging techniques enabled the direct localization and spatial distributions of both apatite crystals and atomic elements (Ca, P) within matrix vesicles and collagen. In longitudinal planes of section, a consistent vectorial gradient of mineralization was observed which started with the first localization of apatite mineral in matrix vesicles; with further development, the mineral spread from the vesicle to the extravesicular interstices and then into the adjacent collagen fibrils. Once intrafibrillar, the mineral was observed to advance both laterally and axially. The association of vesicle/collagen mineral was examined by ESI analysis of Ca and P elemental maps and appeared as a continuum between the vesicles and the adjacent collagen fibrils. Similarly, an intimate spatial relationship was observed between the mineral of vesicles and collagen in transversely cut sections of tendon. The sequential development of this mineralized matrix is discussed in light of matrix vesicle/collagen interactions.

Vesicle- and collagen-mediated calcification in the turkey leg tendon.

The distribution, organization, and orientation of apatitic mineral associated with vesicles and collagen in normal calcifying turkey leg tendon have been examined to determine inorganic-organic interactions in this vertebrate tissue. The study utilized selected area electron diffraction and bright or dark field transmission electron microscopy of thin sections (approximately 70 nm) treated by anhydrous means. Bright field microscopy revealed crystals randomly disposed in radial clusters related to vesicles or highly oriented related to collagen. Dark field images of the crystal c-axis were localized within some hole zone regions of collagen but were generally sparse in vesicles. Images of mineral a,b-axes were associated with vesicles and also appeared in collagen hole zones complementary to those containing c-axis images. Where mineralization of vesicles occurred adjacent to that of collagen, bright and dark field images provided suggestive evidence that the crystals could be contiguous at some sites, but the bulk of extracellular mineralization was spatially discontinuous. The varying disposition of mineral in vesicles and collagen is likely indicative of distinct mechanisms of nucleation in these components, the complementary orientation of mineral c- and a,b-axes in collagen may provide insight into the structure and organization of the protein, and the distribution of vesicle-mediated mineralization appears to contribute to apatite accumulation between collagen fibrils.

Aluminum intoxication in vitamin D-deficient rats: studies of bone aluminum localization and histomorphometry before and after vitamin D repletion.

Aluminum accumulation by both dialysis patients and nonuremic patients, requiring chronic total parenteral nutrition, may be an etiological factor in the development of severe osteomalacia. To study the role of aluminum toxicity in bone, further experiments have been conducted in the nonuremic, vitamin D-deficient rat. Weanling rats were raised on vitamin D-deficient diets, and half received parenteral aluminum (5 mg/wk), for 30 days. In the first experiment low doses of 25-OH cholecalciferol (500 ng/week) were given subcutaneously for a further 30 days. Control rats were maintained on a similar protocol, but were supplemented with cholecalciferol (5 micrograms/week) from the outset until sacrifice at 60 days. In the second experiment a single bolus of cholecalciferol (5 micrograms) was given to study short-term changes in serum biochemistry and bone histology at 96 hr. Quantitative bone histomorphometric analyses of the proximal tibial metaphysis were made in all experimental groups. In the experimental vitamin D-deficient group, with the highest bone aluminum content (as assessed by extraction of whole bone aluminum), X-ray microanalysis was performed to determine the distribution of aluminum in bone tissue and bone cell organelles. The results showed that control rats treated with prolonged aluminum therapy (30 mg over 60 days) had evidence of both reduced osteoid matrix synthesis and mineralization. However, in vitamin D-deficient rats, there was no evidence that aluminum exacerbated the osteomalacic lesion, even though there was histochemical evidence of aluminum deposition at the bone-osteoid interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron microscopic analysis of mineral deposits in the calcifying epiphyseal growth plate.

Early mineral deposits within calcifying rat epiphyseal growth plates were studied by bright field and selected-area dark field electron microscopy, and X-ray microanalysis. These mineral deposits were prepared in situ by high-pressure freezing, freeze substitution, and low-temperature embedding, and were examined in unstained, stained, and ethyleneglycol tetraacetic acid (EGTA)-treated stained thin sections. On unstained sections mineral rods occur within an amorphous density of calcium and phosphorus (CaP). X-ray microanalysis of stained sections reveals that the location of electron-dense deposits does not always correspond to that of the CaP mineral deposits identified in electron microscopic images. Such an analysis showed a depletion of both Ca and P in stained sections at sites corresponding to high levels of these elements in unstained sections. Staining thus demineralizes early deposition sites of CaP; at the same time lead (Pb) and uranium (U) bind to the organic components of the extracellular matrix formerly associated with Ca and P. This substitution phenomenon alters the overall fine structure of mineral sites by depleting the amorphous density of Ca and P, and by creating isolated rodlike structures that have formerly been interpreted as representing hydroxyapatite (HAP) crystals. Selected-area dark field imaging shows nascent sites of HAP crystals to be associated with the limiting membrane of matrix vesicles, but such crystals were undetectable at these sites with conventional bright field images. Dark field imaging also showed that the typical 30-80 nm crystal rods found in calcified cartilage consist of aggregates of HAP crystals.

An electron microscopic and spectroscopic study of murine epiphyseal cartilage: analysis of fine structure and matrix vesicles preserved by slam freezing and freeze substitution.

Newborn mice epiphyseal growth plates were preserved by slam freezing/freeze substitution and examined by conventional electron microscopy, stereopsis, high voltage electron microscopy, and electron spectroscopic imaging (ESI). To illustrate the improved ultrastructure of this cryogenic procedure, conventional, aqueously fixed growth plates were included showing collapsed hypertrophic chondrocytes surrounded by a depleted and condensed extracellular matrix. In contrast, the cryogenically prepared epiphyses contain chondrocytes and extracellular matrix vesicles both in direct contact with proteoglycan filaments retained in an expanded state. ESI is an electron microscopic technique which enables the direct localization of atomic elements superimposed over fine structural details. This technique was used to examine the colocalization of calcium and phosphorus within matrix vesicles and within their associated extracellular environments. Matrix vesicles appeared in three distinct diameter ranges. The integrity of the matrix vesicles was examined at various stages of mineralization and also within the mineralized zone of provisional calcification.

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.

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.

Quantitative spatial distributions of calcium, phosphorus, and sulfur in calcifying epiphysis by high resolution electron spectroscopic imaging.

Electron spectroscopic imaging, a new technique that permits the quantitative detection of the spatial distributions of atomic elements at high resolution, has been applied to the epiphyseal zone of hypertrophy in the mouse for the visualization of calcium, phosphorus, and sulfur. Longitudinally sectioned epiphyseal growth plates reveal a developmental sequence in the longitudinal septum leading from a noncalcified matrix to a calcified matrix. During the early stages of this transition, matrix granules containing highly localized concentrations of P (200-400 atoms/nm2) are found spatially separate from Ca-containing sites. These Ca localizations displayed a concentration range of 20-350 atoms/nm2 and a complete spatial overlap with sulfur. At these sites, S levels range from 10 to 200 atoms/nm2. At a later stage, and therefore more proximal to the zone of provisional calcification, the usual scattered, irregularly shaped mineral deposits are found. These sites contain a virtual superposition of Ca with both P and S. The Ca/P and Ca/S ratios of these mineral deposits are predominantly 1.0 with only minor, locally varying ratios present.