Elemental microanalysis of fibroblasts by a scanning proton microprobe and application to Menkes’ disease

A scanning proton microprobe has been used for the elemental microanalysis of individual fibroblast cells. Both normal fibroblasts and fibroblasts cultured from patients with Menkes' disease, an X-linked genetic disorder known to be associated with defective copper metabolism, were examined by the probe. The cells were cultured on a thin ultra-clean nylon foil and retained on that surface for analysis. The focused high-energy proton beam was used to irradiate selected individual cells and elemental information was derived from X-ray and backscattered proton data. The sensitivity of the scanning proton microprobe to trace concentrations of heavy elements has allowed this elemental information to be used to identify individual cells as being either normal or a Menkes' mutant. The cell identification was based on the application of discriminate analysis to a data set formed from the ratios of copper to each of the macroelements present in the cell. This method of cell identification offers the promise of rapid diagnosis of Menkes' disease.     

Distribution of elements in the lily pollen tube tip,determined with the Oxford scanning proton microprobe

Summary Pollen tubes ofLilium longiflorum were fixed with glutaraldehyde and investigated unsectioned with the Oxford scanning proton microprobe (SPM). Two-dimensional maps which show the distribution and concentration of phosphorus, sulphur, chlorine, potassium, calcium, iron, copper, zinc and arsenic are presented. The maps show that, within the pollen tube tip region, calcium and zinc exhibit relatively steep longitudinal concentration gradients compared to the more flat distributions of phosphorus and sulphur. Chlorine, potassium, iron and copper appear equally distributed along the tube. All elements with the expception of arsenic show the highest concentration within the cell protoplasm and not in the cell wall. Additional signals of arsenic, chlorine and potassium originate from the remaining fixative dried around the tube, containing also the free ions of the cell. The arsenic signals originate exclusively from the buffer used during fixation. The different maps are compared and discussed in relation to their significance to the pollen tubes.Abbreviations EDAX energy dispersive analysis of X-rays - EIXE electron induced X-ray emission - PIXE proton induced X-ray emission - SPM scanning proton microprobe     

Elemental microanalysis of individual blood cells

A scanning proton microprobe was used to study single red blood cells in freeze-dried, whole-blood specimens. Simultaneous collection of PIXE and of forward and backward scattered proton data provided information on the heavier elements (Z>27) and on the organic mass under investigation. The trace elemental spectrum of a red cell was found to be reproducible, and no elemental losses were observed during proton bombardment. The spatial resolution of the probe (1.5 μm for these studies) enabled the red cell’s biconcave disk shape to be visualized in quantitative two- and three-dimensional maps of H, C, P, S, Cl, K, and Fe.     

Some medical applications of the oxford scanning proton microprobe

Results from several medical investigations carried out using the Oxford scanning proton microprobe are presented, including maps of the hepatic copper-distribution in primary biliary cirrhosis and maps of the iron distribution in primary hemochromatosis. Preliminary studies of human nervous tissue reveal that morphologically recognizable structures can be differentiated using precision elemental mapping, and this may lead to a powerful new way of distinguishing subtle perturbations of structure and function. In further prelimianry studies, localized aluminum structures have been observed in Alzheimer’s disease tissue at bulk levels of less than parts per million.     

A proton microprobe scanning across the vertebra of a flat fish,Paralichthys olivaceus

Elemental distributions across the vertebra of a flat fish were determined by a 100×300 μm proton scanning microprobe. By normalizing with proton currents, 22 elements demonstrated characteristic patterns in locality; Si, Ca, Cr, Ga, Sr, and I were homogeneous across the vertebral centrum, whereas concentrations of P, Ti, V, Co, Ni, Cu, Hg, Se, and Br were increasingly higher toward the inside of the centrum. Opposite results were obtained for S and Cl, and especially Fe, which showed gradually lower concentrations toward the center. The K, Mn, and Zn were higher in concentration at both edges, showing a minimum value at the middle of the vertebra centrum, where Pb was at a maximum in its distribution. The fact that such a thin intervertebrale membrane contains relatively large amounts of Cl, K, Ca, Fe, Zn, Pb, Br, and I is interesting.     

Distribution of Elements in Rat Peripheral Axons and Nerve Cell Bodies Determined by X-Ray Microprobe Analysis

X-ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, myelin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellular distribution of elements was clearly shown by color-coded images generated by computer-directed digital x-ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.     

Investigation of P and S Distributions in the Roots of Tagetes patula L. using Micro-PIXE

A proton microprobe for energy-dispersive PIXE-analysis hasbeen used to measure elemental distributions of sulphur andother elements in roots of Tagetes patula L. by making a line-scanalong the diameter of the cross-section. Higher concentrationsof phosphorus and sulphur were found in or near the phloem.In addition the endodermis contained an increased sulphur concentration.The results obtained and future possibilities for this kindof investigation are discussed. Key words: Tagetes patula L., micro-PIXE, sulphur, thiophenes, endodermis, Pratylenchus penctrans (Cobb)     

Demonstration of polar zinc distribution in pollen tubes ofLilium longiflorum with the Heidelberg proton microprobe

Summary The zinc distribution in pollen tubes ofLilium longiflorum was analysed by proton-induced X-ray emission (PIXE) with the Heidelberg proton microprobe. A very high zinc concentration was measured in the pollen tube tip region.On leave from Shanghai Institute of Nuclear Research, Academia Sinica, Shanghai, China     

Distribution of calcium and other elements in cryosectioned Bacillus cereus T spores, determined by high-resolution scanning electron probe x-ray microanalysis.

The distribution of a number of key elements in Bacillus cereus T spores was determined by high-resolution scanning electron probe X-ray microanalysis. To circumvent the redistribution of soluble or weakly bound elements, freeze-dried cryosections of spores, which had been rapidly frozen in 50% aqueous polyvinyl pyrrolidone, were employed. The sections were examined by using a modified Philips EM400 electron microscope fitted with a field emission gun, scanning transmission electron microscopy attachment, and a computer-linked energy-dispersive X-ray microanalysis system. X-ray maps for selected elements and the corresponding electron image were produced simultaneously by scanning the cryosections with a fine electron beam in a raster pattern, using the scanning transmission electron microscopy attachment. The results indicated that almost all of the calcium, magnesium, and manganese, together with most of the phosphorus, was located in the core region. An unexpectedly high concentration of silicon was found in the cortex/coat layer. Granules containing high concentrations of calcium, manganese, and phosphorus were demonstrated in spores containing reduced levels of dipicolinic acid. Spot mode analyses, in which a stationary beam was located over the region of interest in the spore cryosection, confirmed the results obtained with the scanning mode and also provided a more accurate quantitation of the elemental concentrations on a dry weight bases.     

A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger

Although Aspergillus niger is used as a host for heterologous protein production, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a mechanism similar to that in other eukaryotes, but with proteins destined for secretion being directed to the hyphal tip. We report on a method using a glucoamylase: GFP gene fusion which allows us for the first time to monitor, in vivo, protein secretion in A. niger at the single hyphal level. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A. niger glucoamylase (GLA:499). Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A. niger genome. Confocal and fluorescence microscopy revealed that the GLA::GFP fusion protein is fluorescent in A. niger and appears to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence is apparent and immunogold labelling of GFP confirmed that GFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::GFP was detected only in culture filtrates of young mycelia grown in a soya milk medium. The actin inhibitor latrunculin B was used to disrupt the secretion process, and its effects on the distribution of GLA::GFP were monitored.     

Zn对细胞保护作用机理的研究

应用扫描质子微探针和同步辐射ｘ荧光分析技术测定了细胞中元素的分布和组成,为确定Zn是细胞结构成分提供了直接的实验依据.用上述核技术结合有关生化指标,分析测定了正常和损伤细胞（脂质过氧化损伤）中Fe,Zn和丙二醛、SH基含量变化的相互关系.实验结果表明,当细胞发生脂质过氧化损伤时,Fe含量和丙二醛含量同步增高,而Zn含量和SH基量则降低.给细胞补充Zn后,提高了细胞质膜中的Zn含量,SH基量也随之增加,同时丙二醛量降低.提示Zn保护细胞完整性的作用机理之一是控制脂质过氧化作用.Zn可保护膜蛋白的SH基,减少和阻止被Fe所催化的过氧化反应.     

Elemental composition of the human atherosclerotic artery wall

The elemental composition of the human atherosclerotic popliteal artery was examined using the proton-induced X-ray-emission (PIXE) method. The application of a narrow proton beam (3 X 10 micron 2) enabled us to determine not only the concentrations of Cl, K, Ca, Fe, Cu, Zn, Br and Pb, but also their localization in different artery-wall regions. The highest mean concentrations of Cl, K, Zn and Br were found in the tunica media. In the investigated sections the distribution of Ca and Fe varied: sometimes, these elements were prevalent in the tunica intima, whereas in other cases, the highest concentrations were observed in the tunica media or tunica adventitia. The concentration profiles of each element were characterized by many sharp, narrow peaks. The highest concentrations of Ca and Fe showed such high levels that only one explanation is possible, i.e. the presence of crystals. The correlation of Ca peaks with those of Zn and Fe is discussed. The usefulness of the micro-PIXE method for the investigation of biomedical materials is also considered.     

The use of PIXE in experimental studies of the physiology of human skin epidermis

In order to understand the normal and pathological physiologies of the epidermal cells, the simultaneous determination of several elements in the different cellular strata is of crucial importance. In recent years the electron microprobe (EMP) has become an established technique in this field. Its high spatial resolution, in principle, allows measurements of various cell organelles. However, the limited (intrinsic) sensitivity of the EMP represents a serious drawback to the technique. The introduction of the proton microprobe (PMP) has significantly improved the sensitivity, although the ultimate spatial resolution of the PMP is much less than that of the EMP. When studying the elemental profiles in skin epidermis, it is possible to use skin sections with a thickness of the order of 10 μm, then the spatial resolution of the PMP is equal to or better than that of the EMP since the electrons are scattered to a significant degree in the sample. The characteristics of the two methods have been compared by analysis of parallel duplicate freeze-dried sections of normal human skin. The distributions of the elements P, S, Cl, and K, obtained with the two techniques, were in good agreement. In addition, the PMP provided distributions of the important elements Ca, Fe, and Zn. In a recently started study, the useful features of the PMP will be used for studying how efficient a barrier the skin is to nickel and chromate ions. A preliminary experiment has been performed by exposing cadaverous skin, not older than 24-h postmortem, to solutions of the two ions. After an 18-h exposure, samples were prepared by shock-freezing and sectioning. The first results from PMP analysis of these samples demonstrate the presence of a nickel and chromium gradient in the outer strata in the epidermis (mainly stratum corneum). A third experiment deals with the physiology of psoriatic skin. Calcium is an important element in the differentiation. Hence, the higher sensitivity of the PMP has been used in analysis of sections from psoriatic skin epidermis. Preliminary results are presented.     

Changes of elemental concentrations around and on the surface of fowl sperm membrane during maturation in the male reproductive tract and after in vitro storage

X-ray microprobe analysis was performed to investigate the changes of elemental concentrations around or on the membrane of the head, midpiece, and principal piece regions of individual fowl spermatozoa during maturation in the male reproductive tract and after storage in vitro at 4°C. The pattern of change of elemental concentrations during maturation and postejaculation was, in general, similar in the three different subcellular regions; i.e., concentrations of sodium, potassium, chlorine, and calcium decreased gradually during sperm passage through the male reproductive tract and after storage. Phosphorus concentration remained almost constant in the male tract and decreased gradually after storage. In contrast, magnesium, zinc, and copper concentrations showed an interesting pattern: concentrations increased significantly during maturation to a maximum at ejaculation and decreased again after storage. The ratios of sodium to potassium in the midpiece region showed patterns similar to those of magnesium, zinc, and copper concentrations.     

A drop of intracellular pH stimulates citric acid accumulation by some strains of Aspergillus niger

By comparing kinetic parameters of plasma membrane proton pumps from two Aspergillus niger strains, significant differences in specific activities were observed. In low citric acid producing A158 strain the H+ -ATPase activity was about four-fold higher than in a high yielding A60 strain. Previously pH homeostasis was reported in A158 strain while in A60 strain spontaneous drop of intracellular pH was observed. During the growth in the medium with ammonium ions more rapid drop of extracellular pH was recorded with A158 strain and not so fast proton accumulation in the medium with A60 strain, indicating that proton pumps from later strain perhaps can not extrude all the protons that are released in the cytosol after the assimilation of ammonium ions. Vanadium ions were found to be potent inhibitors of both H+ -ATPases. By adding sodium vanadate in millimolar concentrations to the chemically defined medium that induces citric acid accumulation by A. niger, reduced pHi and increased rate of acid production was observed in A158 strain while in A60 strain intracellular pH decreased below 6.5 and concomitantly citric acid overflow was suppressed. The presented results suggest that one of the mechanisms stimulating citric acid accumulation by A. niger could be also a slight cytoplasmic acidification.     

Composition and Distribution of Elements and Ultrastructural Topography of a Human Cardiac Calculus

Trace elements (TEs) may contribute to the formation of calculi or stones or be involved in the aetiopathogenesis of stone diseases. The compositions and spatial distribution of elements from the inner nucleus to outer crust of the cardiac calculus were investigated by energy-dispersive X-ray fluorescence (EDXRF) spectrometer. The surface topograph, distribution map of elements, elemental and chemical compositions were also determined by environmental scanning electron microscope (ESEM)–energy-dispersive X-ray (EDX) analysis. Twenty-five elements were identifiable from 18 positions on the cardiac calculus by EDXRF spectrometer, in which the highest concentrations of toxic TEs (Ni, Pt, Hg, Sn, Pb, W, Au, Al, Si) and higher levels of essential TEs (Ca, Sr, Cr, P) were detected. A moderate positive Pearson’s correlation between TEs concentrations of Mg, Ca or P and location differences from centre to periphery in the cardiac calculus was observed. A positive correlation was also found for Ca/Zn and Ca/Cu, indicating the gradual increase of calcium concentration from inner nucleus to outer crust of cardiac calculus. The drop-like nodules/crystals on the surface of petrous part of cardiac calculus were observed from ESEM analysis. ESEM–EDX analysis determined the calculus to be predominantly composed of calcium hydroxyapatite and cholesterol, as indicated by the petrous surface and drop-like nodules/crystals, respectively. This composition was confirmed using a portable Raman analyser. The spatial distribution analysis indicated a gradual increase in Mg, P and Ca concentrations from the inner nucleus to the outer crust of the cardiac calculus. The major chemical compositions of calcium hydroxyapatite and cholesterol were detected on this cardiac calculus.     

Intramembranaceous ossification analyses by a proton microprobe

The proton induced X-ray emission method in combination with a proton microprobe was applied to study the intramembranaceous ossification. As material sections of mouse embryo skulls from the 17th and 19th day of gestation were used. The morphology of the sample was examined by routine histochemical procedure performed on the sections adjacent to that irradiated by the proton microprobe. The measurements were made in line scan and raster scan mode. The concentrations of P, S, Cl, K, Ca, Fe and Zn were determined at each irradiated point. The average element concentrations were calculated for four parts of each section (bone, cartilage, mesenchymal tissue close to the bone and mesenchymal tissue in other places). The distributions of Ca and P (less markedly than Ca) concentrations almost exclusively correlate with localization of the bone while S, Cl and K concentrations show preference to the cartilage. The amount of inorganic material in flat bones of the 17-day embryo amounts to 14% of the dry mass. The material is characterized by a Ca/P ratio of about 1.6. In the embryo 2 days older the amount of the inorganic phase is practically the same (15%) while the Ca/P ratio approaches 2. This suggests the presence of the precursor phase in the flat bone calcification. It is possible that octacalcium phosphate (Ca/P ratio equals to 1.72) is formed at the onset of the flat bone mineralization which transforms rapidly (in 2 days) to a more stable mineral (defective hydroxyapatite).     

Intramembranaceous ossification analyses by a proton microprobe

Summary The proton induced X-ray emission method in combination with a proton microprobe was applied to study the intramembranaceous ossification. As material sections of mouse embryo skulls from the 17th and 19th day of gestation were used. The morphology of the sample was examined by routine histochemical procedure performed on the sections adjacent to that irradiated by the proton microprobe. The measurements were made in line scan and raster scan mode. The concentrations of P, S, Cl, K, Ca, Fe and Zn were determined at each irradiated point. The average element concentrations were calculated for four parts of each section (bone, cartilage, mesenchymal tissue close to the bone and mesenchymal tissue in other places). The distributions of Ca and P (less markedly than Ca) concentrations almost exclusively correlate with localization of the bone while S, Cl and K concentrations show preference to the cartilage. The amount of inorganic material in flat bones of the 17-day embryo amounts to 14% of the dry mass. The material is characterized by a Ca/P ratio of bout 1.6 In the embryo 2 days older the amount of the inorganic phase is practically the same (15%) while the Ca/P ratio approaches 2. This suggests the presence of the precursor phase in the flat bone calcification. It is possible that octacalcium phosphate (Ca/P ratio equals to 1.72) is formed at the onset of the flat bone mineralization which transforms rapidly (in 2 days) to a more stable mineral (defective hydroxyapatite).