[Biological applications for x-ray microanalysis in electron probe microscopy].
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Electron Probe Microanalysis↗
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Visualization of sulfur-containing components associated with proliferating chondrocytes from rat epiphyseal growth plate cartilage: possible proteoglycan and collagen co-migration.
Electron microscopy of epiphyseal growth plate cartilage from normal 4-5-week-old rats has revealed extensive fibrillar aggregates and globules in the pericellular spaces of proliferating chondrocytes. These cells contained small globules and diffusely coiled, fine filaments located within large, membrane-invested vacuoles. All such structures were observed after a variety of different tissue fixation regimes, including glutaraldehyde, osmium tetroxide, and potassium pyroantimonate. The fibrillar aggregates and globules were often overlapping and intermeshed and extended to 0.5 micron in length from their point of origin at cell membranes. Vacuoles were usually found at the periphery of cells, and some, by membrane fusion with the cell envelope, appeared contiguous with extracellular spaces wherein their contents could be discharged. Fine filaments and globules were occasionally observed in the Golgi complex and cisternae of endoplasmic reticulum of the chondrocytes. Further characterization of the cellular and pericellular components by electron microscopic radioautography, electron probe microanalysis, and electron spectroscopic imaging indicated the presence of sulfur, a result suggesting these aggregates, filaments, and globules in part represent proteoglycans in various stages of synthesis, secretion, and assembly. Additional radioautography utilizing 3H-proline implied that filament bundles are also composed of collagen, a result posing the possibility that this protein and the putative proteoglycans may co-migrate both intracellularly and within pericellular matrices. In extracellular matrices adjacent to cell lacunae, the fibrillar aggregates appeared in close association with typical collagen type II fibrils, an observation providing evidence for proteoglycan-collagen network formation in this region of the rat epiphysis. These microscopic and analytical data in situ would support certain studies in vitro of proteoglycan-collagen type II and IX association and are important in describing the interaction of such cartilage components ultimately involved in matrix formation.
Animals↗
Crystalloid nature of mineral deposits in Bowman's capsule of the human glomerulus. Study by X-ray microanalysis and electron microdiffraction.
Abnormal crystalline mineral deposits are often observed in the basement membrane of Bowman's capsule in the human glomerulus. The nature of these deposits has been the subject of much discussion. By means of electron probe microanalysis and electron diffraction techniques, we have identified these abnormal deposits as whitlockite (beta-calcium-orthophosphate).
Calcium Phosphates↗
Subcellular calcium content in cardiomyopathic hamster hearts in vivo: an electron probe study.
In the Syrian cardiomyopathic hamster heart, abnormal cellular calcium regulation, resulting in cellular calcium overload, is believed to play a role in the pathogenesis of cardiac hypertrophy and failure. Alternatively, the primary abnormality may be coronary vasospasm, resulting in reperfusion-induced necrosis. According to the latter hypothesis, only those cells that suffer an ischemic insult would contain elevated calcium levels. To determine whether a generalized elevation in myocytic calcium exists in myopathic hamster hearts, we measured cellular and subcellular calcium concentrations by electron probe microanalysis in cryosections of 50-day and 96-day myopathic and control hearts, rapidly frozen in vivo. Total calcium content of ventricular homogenates from each group was also measured by atomic absorption spectrophotometry. No significant differences in subcellular calcium were found by electron probe microanalysis among 50-day and 96-day myopathics and their age-matched controls. In 50-day myopathic and control hearts, mitochondrial calcium was 0.7 +/- 0.2 and 0.9 +/- 0.2, respectively, and A-band calcium was 3.0 +/- 0.4 and 2.6 +/- 0.4 mmol calcium/kg dry wt(+/- SEM). Results from 96-day animals were similar. Localized regions of elevated calcium were found only at sites of necrotic foci: in Na+-loaded cells (mitochondria: 4.7 +/- 1.3 (SEM) mmol/kg dry wt), in dying cells (mitochondria: 72 +/- 22 (SEM) mmol/kg dry wt) or as extracellular deposits (7-10 mol/kg dry wt). Total calcium content of hearts from myopathic hamsters, as determined by atomic absorption spectrophotometry, was also 13 times (50-day) and 50 times (96-day) higher than controls. These results demonstrate that there is a marked heterogeneity in cellular calcium content in myopathic hamster hearts, but the data do not support the hypothesis of a generalized cellular calcium overload.
Animals↗
Morphologic features and nuclide composition of infarction-associated cardiac myocyte mineralization in humans.
Low dietary Mg results in Ca loading of cardiac myocytes, which increases the likelihood of myocyte calcification in the event of acute myocardial infarction (AMI), and possibly increases myocyte vulnerability to necrosis. Bloom and Peric-Golia1 previously reported an autopsy study of cases from the Washington, D.C. area (a region with low levels of Mg in the drinking water), demonstrating AMI-associated mineralization in myocytes with histologically normal nuclei and cross striations, as well as in obviously necrotic myocytes. The authors have re-examined mineralized myocytes from the same autopsy material, using electron probe microanalysis, light microscopy, and transmission electron microscopy. Microprobe analysis identified Ca and P as the nuclides composing the inorganic phase of the mineral deposits. Ultrastructurally, all Ca deposits, regardless of size or intracellular location, were composed of aggregates of needlelike hydroxyapatite crystals. The mildest form of intracellular Ca deposition was observed as small Ca deposits limited to some mitochondria of myocytes, which demonstrated intact nuclei and regular sarcomere pattern. More advanced stages of intracellular calcification, in the form of Ca deposits associated with mitochondria, Z-band regions and nuclei, were observed in other myocytes that also retained intact nuclei and sarcomeres. Massive Ca deposits were associated with myocytes which showed morphologic features of advanced necrosis, including loss of nuclei, disruption of sarcomere structure and masses of cellular debris. These observations support the theory originally proposed by Bloom and Peric-Golia1 suggesting that Ca loading of myocytes, possibly related to Mg deficiency in humans, increased vulnerability of the myocytes to subsequent AMI-associated necrosis and dystrophic calcification. In addition, the light microscopic impression of calcification of otherwise normal myocytes is contradicted by the electron microscopic identification of hydroxyapatite crystals free in the sarcoplasm, a condition unlikely to be compatible with viability. Lastly, the fact that all Ca deposits were in the form of hydroxyapatite supports the view that they were formed in a Mg-poor environment, which favors conversion of the more common amorphous form of Ca phosphate into the needlelike crystals of hydroxyapatite.
Calcium↗
Metal binding by intestinal mucus.
Electron probe microanalysis offers distinct advantages for the study of intestinal mucus. This technique permits analysis of metal binding in situ, requires only a small amount of tissue, allows several experiments to be performed with one animal, and can resolve variations in binding that may occur in different portions of the intestine. We have used electron probe microanalysis to examine the metal binding capacity of intestinal mucus in situ. We have exposed portions of excised intestine to various concentrations of several metals, rapidly frozen the tissue and freeze dried it. After anhydrous embedding, thick sections were cut and analyzed on silicon discs or carbon coated copper grids. Qualitative analysis shows two distinctive patterns of distribution. The results of this work show clearly that at least three divalent cations are bound by mucus, that mucus exhibits different affinities for different metals, and that binding of metals is not uniform throughout mucus.
Animals↗
Microanalytical study of thorium 232 deposits in bone marrow and liver.
Analytical microscopy was used to study the distribution and chemical composition of thorium deposits in bone marrow and liver after injection of thorium dioxide and thorium nitrate. Thorotrast (thorium dioxide) was identified as being localized in bone marrow macrophages of a patient who had undergone cerebral arteriography forty two years ago. Large thorotrast deposits were also present in liver cells. We show that non-colloidal thorium (thorium nitrate) injected in rats concentrates in a non soluble form in bone marrow macrophages, hepatocytes and Kupffer cells. These deposits of thorium associated with phosphorus can be explained by the formation of thorium phosphate in lysosomes and we demonstrate that they remain in tissue for a long time. Microanalysis was performed with ion microscopy, and electron probe microanalysis by X ray spectrometry, which can identify and localize thorium and associated elements at cellular or intracellular level.
Animals↗
[Selective localization of neptunium-237 in nuclei of mammalian cells].
After injection in the rat of soluble neptunium salt, the distribution of this element was studied at the subcellular level by electron microscopy and electron probe microanalysis. Abnormal structures have been observed by electron microscopy in the nuclei of hepatocytes, and the same structures have also been observed in the nuclei of the proximal tubules cells of the kidney. These structures are formed of clusters of very small and dense particles, several nanometers in diameter. The clusters are localized in the central part of the nuclei and they are separate from nucleoli and heterochromatin. Electron probe X-ray analysis of this cluster have shown that they contain neptunium associated with phosphorus. In the cell containing neptunium inclusions, other non specific lesions are also observed (nuclear pycnosis, mitochondrial depletion).
Animals↗
[An electron probe x-ray microanalysis of dental alloys].
Electron probe x-ray microanalysis of 3 commercial cobalt-chromium-based alloys, CCA (USSR), Biosil and Vakukast (FRG), has revealed that CCA is initially heterogenic, this being explained, probably, by elevated chromium content. Biosil and Vakukast are similar in composition and represent a matrix with eutectic insertions of intermetallides and a small amount of oxides. Physical and mechanical characteristics of these alloys are described.
Dental Alloys↗
Penetration of ions from silicate cement restorations into Copalite--covered cavity walls.
This study aimed to asses the effect of silicate cement on Copalite -covered cavity walls in extracted human teeth. Class V cavities were prepared in 24 premolars and filled with silicate cement (Bio-Trey). Four cavities were unlined, the rest of the cavities were lined with 1 or 2 layers of Copalite before insertion of the restorations. After 6 months, 70-100 microns thick longitudinal sections of the teeth were studied by polarized light microscopy, microradiography and electron probe microanalysis. When imbibed in water or quinoline, a subsurface zone of altered birefringence was noticed in nearly all cavity walls. Nearly half of the cavity walls in the experimental groups showed a surface zone of increased radiopacity. In a few instances a subsurface radiolucent zone was present. By electron probe microanalysis F (0,4-3% by weight), Zn (1-4%) and Al (0,2-6%) were measured in the outer 10-60 microns of the cavity walls. The study shows that even with a double layer of Copalite, known to prevent microleakage, a desirable uptake of F and Al from silicate restorations into cavity walls can take place. Copalite does not prevent a phosphoric acid effect on the cavity walls.
Aluminum↗
Electronic core level microanalyses and microcopies in multipurpose apparatus.
Using an instrument equipped with two electron guns, an electron analyzer, and a Si(Li) diode detector, we developed microanalytical techniques based on inner-shell electron excitations by incident electrons and X-rays, that is, electron energy-loss spectroscopy (EELS) in the reflection mode; electron probe microanalysis (EPMA) and X-ray appearance potential spectroscopy (XAPS); electron-induced Auger electron spectroscopy (e-AES); X-ray photoelectron spectroscopy (XPS), X-ray absorption spectroscopy (XAS); X-ray induced AES (XAES), X-ray fluorescence analysis (XRF), and scanning X-ray radiography (SXR). The corresponding characteristic images (including X-ray microradiography and X-ray photoelectron microscopy) were obtained in the scanning mode. The principle of the apparatus is described. Each spectroscopy and microscopy is illustrated by an example. Their performance and limits are discussed.
Electron Probe Microanalysis↗
Postvaccinal sarcomas in the cat: epidemiology and electron probe microanalytical identification of aluminum.
An increase in fibrosarcomas in a biopsy population of cats in the Pennsylvania area appears to be related to the increased vaccination of cats following enactment of a mandatory rabies vaccination law. The majority of fibrosarcomas arose in sites routinely used by veterinarians for vaccination, and 42 of 198 tumors were surrounded by lymphocytes and macrophages containing foreign material identical to that previously described in postvaccinal inflammatory injection site reactions. Some of the vaccines used have aluminum-based adjuvants, and macrophages surrounding three tumors contained aluminum oxide identified by electron probe microanalysis and imaged by energy-filtered electron microscopy. Persistence of inflammatory and immunological reactions associated with aluminum may predispose the cat to a derangement of its fibrous connective tissue repair response, leading to neoplasia.
Aluminum↗
Calcium-containing structures in vertebrate glial cells. Ultrastructural and microprobe analysis.
Electron probe microanalysis has revealed that vesicular or cisternal structures containing electron-dense material in frog ependymal glial cells contain deposits of calcium and phosphorus. The so-called "osmiophilic particles" in human astrocytes also contain calcium. It is suggested that these organelles are storage sites of calcium.
Animals↗
Absence of aluminium in Alzheimer's disease brain tissue: electron microprobe and ion microprobe studies.
Brain tissue from the frontal cortex and hippocampal formation, taken at autopsy or biopsy from 7 patients with Alzheimer's disease, was studied by two methods of microanalysis. One case of Down's syndrome was also studied. Electron probe microanalysis of the frontal cortex and Ammon's horn of the hippocampus showed no aluminium in the various cell organelles, especially in the lysosomes, although some aluminium was found in a few contaminating dusts. Ion microscopy, a method of extremely high sensitivity, also showed the absence of an aluminium signal.
Adult↗
Pulmonary mineral dust. A study of ninety patients by electron microscopy, electron microanalysis, and electron microdiffraction.
The results of a study of 90 patients are presented. Intrapulmonary mineral deposits were characterized by electron diffraction and electron probe microanalysis. Using this method, pneumoconioses may be distinguidhed from other pneumopathies. In cases of pneumoconiosis, there exists a specific relationship between the etiology of the dust exposure and the crystallographic characteristics of the intrapulmonary deposits. The nature of the deposits may be indicative of a specific type of pneumoconiosis. This method is particularly useful in differentiating between asbestos bodies and ferruginous bodies. The value of the method in general and its importance in the study of pneumoconiosis are discussed.
Asbestos↗
X-ray analysis of pathological calcifications including urinary stones.
Electron-probe microanalysis (EPM) is an ideal technique with which to study biological calcification. It is particularly effective in identification of crystalline or non-crystalline deposits of minute size in tissues, and in detecting artifacts which may occur during tissue processing. Human aorta, aortic valves, tumours, joint fluid, and calculus specimens were analyzed via scanning electron microscopy (SEM), EPM, transmission electron microscopy (TEM), and selected-area electron diffraction (ED). Crystals found in the specimens were definitively identified by combined SEM-EPM. It is apparent that EPM is an invaluable tool that will potentially improve the acuity of 'in house' laboratory diagnosis of many pathological calcifications.
Aorta↗
Collagen expression, ultrastructural assembly, and mineralization in cultures of chicken embryo osteoblasts.
A newly defined chick calvariae osteoblast culture system that undergoes a temporal sequence of differentiation of the osteoblast phenotype with subsequent mineralization (Gerstenfeld, L. C., S. Chipman, J. Glowacki, and J. B. Lian. 1987. Dev. Biol. 122:49-60) has been examined for the regulation of collagen synthesis, ultrastructural organization of collagen fibrils, and extracellular matrix mineralization. Collagen gene expression, protein synthesis, processing, and accumulation were studied in this system over a 30-d period. Steady state mRNA levels for pro alpha 1(I) and pro alpha 2 collagen and total collagen synthesis increased 1.2- and 1.8-fold, respectively, between days 3 and 12. Thereafter, total collagen synthesis decreased 10-fold while mRNA levels decreased 2.5-fold. In contrast to the decreasing protein synthesis after day 12, total accumulated collagen in the cell layers increased sixfold from day 12 to 30. Examination of the kinetics of procollagen processing demonstrated that there was a sixfold increase in the rate of procollagen conversion to alpha chains from days 3 to 30 and the newly synthesized collagen was more efficiently incorporated into the extracellular matrix at later culture times. The macrostructural assembly of collagen and its relationship to culture mineralization were also examined. High voltage electron microscopy demonstrated that culture cell layers were three to four cells thick. Each cell layer was associated with a layer of well developed collagen fibrils orthogonally arranged with respect to adjacent layers. Fibrils had distinct 64-70-nm periodicity typical of type I collagen. Electron opaque areas found principally associated with the deepest layers of the fibrils consisted of calcium and phosphorus determined by electron probe microanalysis and were identified by electron diffraction as a very poorly crystalline hydroxyapatite mineral phase. These data demonstrate for the first time that cultured osteoblasts are capable of assembling their collagen fibrils into a bone-specific macrostructure which mineralizes in a manner similar to that characterized in vivo. Further, this matrix maturation may influence the processing kinetics of the collagen molecule.
Animals↗
Calcification of alpha-elastin coacervates: a bulk property of elastin.
Scanning electron microscopy and electron probe microanalysis studies are reported on thin sections of calcified coacervates of alpha-elastin. It is found that the capacity of elastin coacervates to initiate calcification is a bulk property of the coacervate and not limited to the serum-coacervate interface, that the calcium phosphate deposits act to bind the protein units together and slow the dissolution and spreading of the coacervate as it floats on an airwater interface, and that, within the limits of detectability, there is no involvement of sulfur. As the charged groups of alpha-elastin had been blocked, the initiation of deposition is due to neutral sites in the protein which are tightly bound to the calcium phosphate deposits.
Animals↗