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

Abstract

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.A fundamcntal problem in the study of calcifying tissues is the identification and characterization of the site of initial mineralization-the nucleation site. The organic matrices of cartilage, bone, and dentin possess common structural components although organized in different characteristic fashions. From ultrastructural and biochemical studies, various components of these calcifying matrices and their parenchyma have been implicated in the role of nucleation-e.g., collagen fibers (1), matrix vesicles (2-4), mitochondria (5), y-carboxyglutamic acid (6), lipids, and Ca-phospholipid-PO4 complexes (7,8). Of these components, various ones may interact in concert to initiate nucleation or to control it in some way. However, the exact mechanism of matrix-mediated mineralization remains controversial.In order to assist conventional microscopic imaging of the dynamic events of calcification, many techniques have been used: pyroantimonate staining for Ca (9, 10), electron probe x-ray microanalysis for detection of elements (2, 11), cationic dyes for staining proteoglycans (12), specific immunofluorescence to localize both monomer core protein and link proteins of proteoglycans (13), and ultracryomicrotomy for the increased preservation of matrix ground substance (2,14). These techniques have helped greatly in establishing the present state of knowledge. However, the techniques have limited the definition of a nucleation site to a Ca-containing mineral deposit with thousands of Ca atoms.In the work reported here, a new technique known as electron spectroscopic imaging was used to study the quantitative distribution of Ca, P, and S in the epiphyseal growth plate. This technique enables the direct visualization of elements in entire images with a spatial resolution as fine as 0.3-0.5 nm and with a minimum detection level as low as 2 X 10-21 g (or 50 atoms for P) (15, 16). Compared ...

Cartilage Calcification Studied by Proton Nuclear Magnetic Resonance Microscopy

“…The latter elements arise most likely from the presence of sulfated PGs and sodium counterions in the tissue. (29) Proton NMR microscopy Signals unique to NMR microscopy were measured for mineralizing bioreactors to compare with data obtained from histology and analytical electron microscopic methods. A series of representative NMR images is shown in Fig.…”

Quantitative energy-filtered image analysis in cytochemistry

“…The recently introduced energy-filtered electron microscopy (EFEM) [10][11][12]25,30,32] certainly has not yet proved to overcome all mentioned limitations, but its electron-spectroscopical imaging potentials [25,26] prompted us to investigate its application for a morphometric analysis using: (1) ESI/TEM images, (2) EELS/TEM elemental distribution images, and (3) ESI/TEM and EELS/TEM image integration.…”

“…De-* To whom correspondence should be addressed. dicated STEM/EELS instruments have analyzed the chemical analytical potentials of this combination in biological and non-biological materials [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]27]. The formation of elemental distribution images has been described for both combinations [5,8,13,14,17].…”

Eliminating Exposure to Aqueous Solvents Is Necessary for the Early Detection and Ultrastructural Elemental Analysis of Sites of Calcium and Phosphorus Enrichment in Mineralizing UMR106-01 Osteoblastic Cultures

“…In those studies epiphyseal growth plates revealed a developmental sequence leading from a noncalcified matrix to a calcified matrix wherein, in the initial stages, matrix granules enriched predominantly in P were distributed separately from Ca-containing sites. At a later stage of mineralization, both calcium and phosphorus were found to instead colocalize with irregularly shaped mineral deposits in the zone of provisional calcification [Arsenault and Ottensmeyer, 1983]. Interestingly, Arsenault and Ottensmeyer [1983] also detected initial elevated focal concentrations of phosphorus in the early stages of intramembranous bone mineralization which were separate from those of calcium [Arsenault and Ottensmeyer, 1984].…”

“…50 atoms [Simon, 1987]. It was developed by Ottensmeyer [1982] to image calcium, sulfate, and/or phosphorus atoms in calcifying cartilage and bone [Arsenault and Ottensmeyer, 1983]. This method has subsequently been used to characterize the calcification of dentine [Plate et al, 1992] as well as the mineralization of osteoblastic cells and chondrocytes in culture [Bordat et al, 1998;Rohde and Mayer, 2007].…”