Visualization and analysis of unfolded nucleosomes associated with transcribing chromatin.

We have characterized the structure of transcriptionally active nucleosome subunits using electron spectroscopic imaging. Individual nucleosomes were analyzed in terms of total mass, DNA and protein content, while the ensemble of images of active nucleosomes was used to calculate a three-dimensional reconstruction. Transcriptionally active nucleosomes were separated from inactive nucleosomes by mercury-affinity chromatography thus making it possible to compare their structures. The chromatographic results combined with electron spectroscopic imaging confirm that active nucleosomes unfold to form extended U-shaped particles. Phosphorus mapping indicated that the nucleosomal DNA also underwent a conformational change consistent with particle unfolding. The three-dimensional structure of the Hg-affinity purified nucleosomes determined using quaternion-assisted angular reconstitution methods unites and resolves the different electron microscopic views of the particle and is concordant with a sulphydryl-exposing disruption of the H3-H4 tetramer.

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.

Experimental ionization cross-sections of phosphorus and calcium by electron spectroscopic imaging.

The absolute partial electron scattering cross-section for the phosphorus L2,3-shell ionization was measured by electron spectroscopic imaging using poliovirus as a primary standard. The equivalent calcium cross-section was obtained in relation to phosphorus using the stoichiometric ratio for these two elements in hydroxyapatite, Ca10(PO4)6(OH)2. At 80 keV, the partial cross-section of phosphorus was 2.26 x 10(-20) and 2.68 x 10(-20) cm2/atom for poliovirus and hydroxyapatite, respectively, at 150 eV loss for a 15-eV energy window and an acceptance angle of 15 mrad. Under the same conditions the calcium cross-section was 0.49 x 10(-20) cm2/atom at 360 eV loss. The experimental values are slightly higher than the theoretical cross-sections calculated either by hydrogenic or Hartree-Slater approaches.

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.

Automatic selection of molecular images from dark field electron micrographs.

A method is described which can be used objectively to select putative molecular images from dark field electron micrographs of unstained molecules. The only characteristic of the molecule required for automatic selection is an estimate of molecular weight. Structures are selected from micrographs by a series of steps including: low pass filtering, edge detection and mass determination. The procedure is shown to be reliable for images with signal-to-noise ratios of at least 4.0. Moreover, the method is insensitive to both the shape and the number of molecules in the image. Five different molecules with molecular weights between MW 330,000 and MW 4000 are successfully selected from low dose STEM and high dose tilt beam dark field electron micrographs.

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.

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.

DNA organization in nucleosomes.

A new technique known as electron spectroscopic imaging has allowed the direct visualization of DNA within the nucleosomes of chromatin. The results presented here confirm the model which suggests that approximately two supercoil turns of DNA are wound about the nucleosome core. The structure of nucleosomes from putative transcriptionally active genes, fractionated by preferential sensitivity to DNAase II and solubility in 2 mM MgCl2, has been examined using both dark field electron microscopy and electron spectroscopic imaging. Oligomeric strands of nucleosomes in this fraction have a less distinct beaded appearance than those of bulk chromatin. The phosphorus distribution in this chromatin suggests that the DNA has a less recognizable organization, lacking a two-turn supercoil per subunit. The unique appearance of this fraction in 30 mM NaCl is reversibly changed to the classical beaded appearance when dialyzed into 0.4 M NaCl.

The correlation between bismuth and uranyl staining and phosphorus content of intracellular structures as determined by electron spectroscopic imaging.

Four groups of intracellular structures can be recognized according to bismuth and uranyl staining and phosphorus content. (1) Those which contain phosphorus and stain strongly with uranyl acetate but not with bismuth (ribosomes, heterochromatin and mature ribosomal precursor granules), presumably because of their nucleic acid content. (2) Those which contain phosphorus and stain with uranyl acetate and bismuth (interchromatin granules, immature ribosomal precursor granules and mitochondrial granules), presumably because at least some of their phosphate is available to react with bismuth. (3) Those which contain little phosphorus but which stain strongly with bismuth and weakly with uranyl acetate (Golgi complex beads), perhaps because some ligand in addition to phosphate reacts with bismuth, and (4) those which do not contain phosphorus and stain with neither uranyl acetate nor bismuth (portasomes). Uranyl staining correlates strongly with the phosphorus content of nucleic acids, proteins and inorganic deposits. Bismuth will stain some phosphorylated molecules but not all. Thus only some phosphates stain with bismuth.

High resolution microanalysis for phosphorus in Golgi complex beads of insect fat body tissue by electron spectroscopic imaging.

Golgi complex beads are 10 nm particles arranged in rings on the smooth forming face of the Golgi complex that stain specifically with bismuth in arthropod cells. In vitro experiments with biological molecules spotted on to cellulose acetate strips indicated that bismuth bound to the beads through phosphate groups. We could detect a weak phosphorus signal from the beads using a new technique called electron spectroscopic imaging that is capable of very high spatial resolution (0.3-0.5 nm) and sensitivity (50 atoms of phosphorus). Detection was not obscured by tissue staining with bismuth or uranyl acetate of by using an inorganic buffer (Na cacodylate). Localization of phosphorus was greatly improved by using colour-enhanced computer pictures of the electron spectroscopic images and quantitating the images. The results indicate that the phosphorus content of the beads is large enough to account for their bismuth reactivity.

Spatial resolution and detection sensitivity in microanalysis by electron loss selected imaging.

An electron energy filter of the Castaing-Henry type in a high resolution transmission electron microscopy was tested for sensitivity and spatial resolution at a specific electron energy loss in energy selected images of a murine leukaemia virus. Electron spectroscopic images of phosphorus within the virus membrane bilayer demonstrated a best spatial resolution between 0.3 and 0.5 nm and a sensitivity of 2 X 10(-21) g.

Phosphorus distribution in the nucleosome.

The spatial distribution of phosphorus within active fraction nucleosomes reveals that the path of the DNA is consistent with one and three-fourths turns of DNA supercoiled around the outside of the protein core. This phosphorus distribution, obtained with an imaging electron spectrometer in a conventional transmission electron microscope, simultaneously establishes new limits of sensitivity for elemental microanalysis.

Visualization of single heavy atoms by dark field electron microscopy.

Dark field electronmicrographs of atoms of palladium, iodine, platinum, osmium, and uranium in model compounds have been obtained. Statistical analyses and a series of blind tests demonstrate the validity of the results. Moreover, optical density measurements of the images indicate that the observed relative scattering cross-sections of these atoms agree well with the theoretical cross-sections calculated from a Thomas-Fermi-Dirac model of the atom.