Quantitative Electron Probe X-ray Microanalysis Research Articles

Elemental microanalysis of organelles in proximal tubules. I. Alterations in transport and metabolism.

Oxygen deprivation to the kidney causes a multifactorial series of morphological, physiological, and biochemical alterations that occur as a function of time. One of the earliest events involves significant changes in the cellular contents of the physiologically important elements (ions) Na and K. Controversy exists as to the nature of changes in the content of the regulatory ion Ca, in either its free or bound form, and much less is known regarding in situ distribution and amounts of other elements such as Mg, P, S, and Cl during physiological or pathophysiological states. The objective of these studies was to evaluate element compartmentation in proximal renal tubules by using quantitative electron probe x-ray microanalysis, during specific conditions which are at least partially manifested during oxygen deprivation. Cells from control proximal tubule suspensions were compared with those exposed to (1) ouabain, to inhibit (Na+, K+)-ATPase; (2) mitochondrial uncouplers, to rapidly deplete ATP content; or (3) calcium ionophores, to cause a rapid elevation in cytoplasmic free calcium. In parallel with electron probe x-ray microanalysis imaging of subcellular elemental content, total cell potassium and ATP contents, enzyme release, oxygen consumption, cytoplasmic free calcium levels, and ultrastructural alterations were assessed. Results indicated that ATP depletion was, in the short term, more deleterious to renal proximal tubules than any of the tested ionic alterations. Intracellular organelles including mitochondria and nuclei appeared to be readily permeable to Na, K, and Cl, altering their concentrations of these ions in parallel with cytoplasmic concentrations. Lysosomes exhibited evidence of Cl accumulation, consistent with an inwardly directed proton ATPase with accompanying Cl transport. Whereas in the cytoplasm Na, K and Cl appeared to be mostly free, a large fraction of these ions within intracellular organelles seemed bound.

Structural and elemental characterization of heart cells grown in a collagen matrix

A novel preparation of spontaneously contracting heart cells embedded in a collagen strand provides an ideal experimental system for correlative structure-function experiments that utilize the techniques of electron microscopy, quantitative electron probe x-ray microanalysis (EPXMA) and imaging. Heart cells grown within the strand for 1 day possess the subcellular content and distribution of physiologically relevant elements—Na, Mg, P, 5, Cl, K, and Ca—found in intact heart cell preparations. The presence of junctional specializations between, and organized myofibrils within, the majority of cells after 1 day in culture also establishes that the collagen matrix promotes vigorous cell development as well as maintains physiological integrity. EPXMA, combined with ultrastructural analyses, provides elemental content data on a cell-by-cell basis. In studies presented here, viable cells, comprising over 80% of the strand cell population, could be distinguished easily from those which had been functionally compromised, not only by aberrant structure but also by altered subcellular compartmentation of Na, K, Cl, and Ca. Within individual viable cells, compartmental differences in element content were notable especially between mitochondria and cytoplasm. However, nuclear euchromatin, but not heterochromatin, appeared approximately identical to cytoplasm in elemental and water content. In such cells, the cytoplasmic K:Na ratio was maintained at a high level (∼15:1). The results with respect to K, Na, and other elements demonstrated the integrity of membrane transport mechanisms regulating the movement and distribution of ions and the maintenance of ionic homeostasis in cells of the strand preparation.

Electron-probe x-ray microanalysis studies on the intracellular calcium translocation during the contraction-relaxation cycle in the fish swimbladder muscle

The swimbladder muscle of a teleost fish,Sebasticus marmoratus, contracts rapidly to produce sounds for communication and contains extremely well developed sarcoplasmic reticulum (SR), providing a material suitable for studying the intracellular Ca translocation during the contraction-relaxation cycle. We examined the change of Ca distribution along the sarcomere at various states of the fibers during the contractionrelaxation cycle in the swimbladder muscle by means of quantitative electron probe X-ray microanalysis of cryosections.The muscle fibers were rapidly frozen at rest, during sustained contraction and at 0.1 and 1.0 sec after the onset of relaxation with a dual-jet liquid propane freezing device. Cryosections (200 nm thick) were cut from the middle portion of the frozen fibers at -110°C on a LKB NOVA cryoultramicrotome, and placed on thin carbon supporting films on Nigrids. Then the cryosections were freeze-dried at 10-6torr and below -80°C. Electron probe X-ray microanalysis was performed on a liquid N2-cooled Be-stage at -130°C in a JEOL 2000FX electron microscope operated at 80 kV and equipped with a Tracor Northern TN5500 energy dispersive X-ray microanalyzer.

Atrial specific granules: The intracellular calcium store

It is well known that certain organells in cytosol contain high calcium concentration and they are named intracellular Ca stores. These stores regulate the intracellular Ca concentration. The present project aims at exploring whether the release of atrial natriuretic peptide (ANP) from the atrial specific granules(ASG) is dependent on the release of calcium from the ASG. For this purpose, we have to know first whether the ASG contain high Ca concentration. In this work, positive results were obtained by quantitative electron probe x ray microanalysis (EPMA) and Ca-ATPase cytochemical localization.Adult female Sprague Dawley rats were used. The auricle samples were prepared in five different procedures: 1 )preparation of routine ultrathin sections; 2)ethanolic phosphotungstic acid(EPTA) staining ultrathin sections for ASG showing; [1] 3) cytochemical localization of Ca-ATPase ;[2] 4) preparation of ultrathin cryosections of fresh rapid frozen auricles; [3] 5) quantitative EPMA with Link AN10000 and Link Quantem/FLS quantitative analysis software.

Comparison of quantitative x-ray imaging and raster probing in heart cells exposed to sodium-free media

Quantitative biological electron probe x-ray microanalysis (EPXMA) ideally makes use of two approaches in examination of samples: static raster probing and quantitative x-ray imaging. Efficient use of expensive microanalytical equipment requires intelligent decisions on the appropriate strategy for optimizing data collection. A low resolution, short dwell time map (for example, 64 × 64 pixels by 2 seconds) may survey one to several cells at low magnification and provide adequate statistics for most structures and elements. Elements, notably Ca, present in low concentrations within small regions may require the acquisition equivalent of long static raster probes, increased numbers of images from low resolution maps, higher resolution maps, longer dwell time maps, or a combination of these options.We examined raster probe and mapping results in cultured heart cells. In these cells EXPMA has been demonstrated to be an appropriate tool to observe the results of perturbation of the Na-Ca gradient, brought about by incubation of cells in low Na or Na-free media.

Responses of the hepatopancreatic ‘B’ cells of a terrestrial isopod,Oniscus asellus, to metals accumulated from a contaminated habitat: A morphometric analysis

The four-lobed hepatopancreas of terrestrial isopods is a highly specialized enzyme secretory, digestive and absorptive region of the midgut. The walls of these blindending tubes are composed of two distinctive epithelial cell types: large, lipid-rich B-cells projecting into the lumen; and small S cells containg (Cu+S)-rich granules. This study investigated the possible stressful effects of accumulated Pb and Zn on the metabolically highly responsive, B-cells. Three major objectives were pursued. First, quantitative electron probe X-ray microanalysis (EPXMA) was employed to determine whether the accumulation of Pb and Zn changes the Fe concentration within individual Fe-granules. Second, simple morphometric techniques were applied to transmission electron micrographs to determine whether competition between essential (Fe) and pollutant (Pb,Zn) metals for binding ligands resulted in an increased granule volume per individual cell. Third, to measure the effect of Pb and Zn accumulation of the amount of stored lipid, in the form of discrete droplets, within the B-cells.

Maintenance of Low Cl− Concentrations in Mesophyll Cells of Leaf Blades of Barley Seedlings Exposed to Salt Stress

The concentrations of vacuolar Na(+) and Cl(-) in the epidermal and mesophyll cells of the leaf blade and sheath of Hordeum vulgare seedlings (cv California Mariout and Clipper) were measured by means of quantitative electron probe x-ray microanalysis. A preferential accumulation of Cl(-) in vacuoles of epidermal cells in both blade and sheath and a low level in mesophyll cells of the blade were evident in plants grown in full strength Johnson solution. The concentration of Cl(-) in the mesophyll cells of the blade remained at a low level after exposure to 50 or 100 millimolar NaCl for 1 day or to 50 millimolar for 4 days, while at the same time the concentration of Cl(-) in the epidermis and mesophyll of the sheath showed a dramatic increase. Clipper generally contained more Cl(-) in the mesophyll cells of the blade than California Mariout. A greater accumulation of Na(+) in the mesophyll of the sheath relative to that of the blade was only apparent after treatment with 100 millimolar NaCl for 1 day or 50 millimolar for 4 days. These results confirm the suggestion that sheath tissue is capable of accumulating excess Cl(-) (and to a lesser extent Na(+)) and suggest that the site of regulation of Cl(-) concentration in the barley leaf is located in the mesophyll cells of the blade.

Zinc sequestration by earthworm (Annelida: Oligochaeta) chloragocytes

The elemental compositions of chloragosome "granules" in the earthworm Lumbricus rubellus living in non-polluted and heavily Zn-polluted soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. The in vivo accumulation of Zn by the chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Zn was apparently bound by at least two ligand pools (Pool 1 = uncharacterised; Pool 2 = P-containing ligands, binding approximately 45% and 55% of the Zn, respectively) in the "control" chloragosomes. In Zn-contaminated chloragosomes, most (approximately 70%) was bound by P-containing ligand(s) but some (less than 1%) was also bound by S-containing ligands. It is suggested that the sequestration of Zn in chloragosomes results in the detoxification of the metal by accumulative immobilisation.

The effect of lead incorporation on the elemental composition of earthworm (Annelida, Oligochaeta) chloragosome granules.

The elemental compositions of chloragosome 'granules' in the earthworm Lumbricus rubellus living in non-polluted (Dinas Powys) and heavily Pb-polluted (Wemyss) soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. High Pb concentrations were found in chloragosomes of Wemyss animals; Pb was not detected in chloragosomes of Dinas Powys animals. Partial correlation and regression analysis indicated that the in vivo accumulation of Pb by chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Pb is bound by P-containing ligand(s) in the chloragosome matrix. The sequestration of Pb by chloragosomes results in the detoxification of the metal by accumulative immobilization.

Electron-microscopic and microprobe analyses on the pigmented and unpigmented enamel of Sorex (Insectivora).

Sorex belongs to the Insectivora and has a pigmented tooth enamel due to iron. The pigmented enamel (PE) has a mean Ca/P weight ratio, analyzed by quantitative electronprobe X-ray microanalysis, of about 1.9 (mean molar Ca/P ratio 1.46), and the unpigmented enamel (UE) a Ca/P weight ratio of about 2.0 (mean molar Ca/P ratio 1.59). The PE has a higher iron content (with a value of about 8%) than the UE, as shown by microanalysis of ultrathin sections. Laser microprobe mass analysis (LAMMA) has shown that the carbonate content in the UE is higher than in the PE. In the LAMMA spectrum of the negatively charged ions the carbonate lines could be compared directly with those of negatively charged iron ions. The pigmentation is associated with a low Ca/P ratio but may transfer mechanical strength and acid resistance strength to the PE.

The use of backscattered electrons in analytical electron microscopy for the measurement of the mass of individual rat blood platelets.

The backscattered electron signal, generated in individual cells, has been used to measure the dry mass of these cells. Absolute mass values were obtained by comparing the backscattered electron signals of cells to the signals of polystyrene-latex spheres of known mass. The technique was carried out in an automated analytical scanning transmission electron microscope and applied to rat blood platelets. The resulting mass distributions agreed well with the distribution measured with a method that uses the transmitted electron signal by means of densitometric analysis of electrographs. Also the range of masses was in agreement with values deduced from data in the literature. The fully automated technique has the advantage that it is direct, fast, and that thicker specimens can be measured than is possible using the transmitted electron signal. The method is intended for use in combination with quantitative electron probe X-ray microanalysis and is then able to produce elemental mass fractions of biological specimens at the subcellular level.

Heterogeneity of calcium compartmentation: electron probe analysis of renal tubules.

The objective of this study has been to determine the intracellular localization of calcium in cryofixed, cryosectioned suspensions of kidney proximal tubules using quantitative electron probe X-ray microanalysis. Two populations of cells have been identified: 1) "Viable" cells, representing the majority of cells probed, are defined by their relatively normal K/Na concentration ratio of approximately 4:1. Their measured Ca content is 4.1 +/- 1.4 (SEM) mmol/kg dry wt in the cytoplasm and 3.1 +/- 1.1 mmol/kg dry wt in the mitochondria, or an average cell calcium content of approximately 3.8 mmol/kg dry wt. 2) "Nonviable" cells, defined by the presence of dense inclusions in their mitochondria and a K/Na concentration ratio of approximately 1. The Ca content is 15 +/- 2 mmol/kg dry wt in the cytoplasm and 685 +/- 139 mmol/kg dry wt in the mitochondria of such cells. Assuming 25 to 30% of the cell volume is mitochondrial, the overall calcium content of such nonviable cells is approximately 210 mmol/kg dry wt. The presence of these inclusions in 4 to 5% of the cells would account for the average total Ca content measured in perchloric acid extracts of isolated proximal tubule suspensions (approximately equal to 18 nmol/mg protein or 12.6 mmol/kg dry wt). Whole kidney tissues display a large variability in total Ca content (4.5 to 18 nmol/mg protein, or 3.4 to 13.5 mmol/kg dry wt), which could be accounted for by inclusions in 0 to 4% of the cells. The electron probe X-ray microanalysis (EPXMA) data conclusively demonstrate that the in situ mitochondrial Ca content of viable cells from the kidney proximal tubule is low and support the idea that mitochondrial Ca may regulate dehydrogenase activity but probably does not normally control cytosolic free Ca.

Quantitative electron probe x-ray microanalysis and densitometric mass determination of individual rat blood platelets.

An improved method for quantitative electron probe X-ray microanalysis of thin biological specimens, introduced recently, has been applied to the elemental analysis of rat blood platelets. The method uses the X-ray signal to quantify the elemental content of an object and electron scattering to determine the total dry mass of the object. Along with the dry mass distribution, data were obtained on the content and mass fraction of calcium, magnesium, and sulfur in 31 individual platelets. The mean platelet dry mass was found to be 985 fg. The mean Ca, Mg, and S contents were 1.80, 3.41, and 18.3 fg, with mean dry mass fractions of 0.195, 0.396, and 1.96%, respectively. Furthermore, these elements appear to be unevenly distributed among the platelet population.

Ultrastructural localization of zinc and calcium within the granules of rat Paneth cells.

Zinc (up to 300 mmol/kg dry weight) and calcium (up to 350 mmol/kg dry weight) were demonstrated in the granules of Paneth cells from the ileum of adult rats by quantitative electron probe X-ray microanalysis of freeze-dried frozen sections. In resin-embedded samples zinc was also localized by histochemical techniques, the validity of which were subsequently evaluated by X-ray microanalysis. The concentration of zinc was highest in granules towards the apex of the cell. Large differences were found between the zinc levels of granules from rats receiving different batches of the same stock laboratory diet. These differences were eliminated by both oral and intraperitoneal administration of supplementary zinc.

Quantitative electron probe x-ray microanalysis of thin films

Quantitative electron probe x-ray microanalysis of thin films

Quantitative electron probe X-ray microanalysis of thin objects using an independent mass determination method

Elemental mass fraction determinations on thin biological specimens by quantitative electron probe X-ray microanalysis commonly use a total mass estimation based on registration of X-ray continuum. We have tested whether the accurary of such determinations on individual biological objects can be improved when a densitometric method for mass determination is used. Air-dried femtoliter droplets with dry masses in the order of 10 −16 kg were used as model objects. An improvement of over two-fold upon the continuum method could be achieved.