Reduction of Potassium in Kidney Proximal Tubules to Aid in Electron Probe X-Ray Microanalysis of Calcium

1985 ◽  
Vol 43 ◽  
pp. 474-475
Author(s):  
A. LeFurgey ◽  
P. Ingram ◽  
L.J. Mandel
Keyword(s):  
Smooth Muscle ◽  
Proximal Tubule ◽  
Electron Probe ◽  
Clinical Applications ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Target Cells ◽  
X Ray ◽  
Freeze Dried

For quantitative determination of subcellular Ca distribution by electron probe x-ray microanalysis, decreasing (and/or eliminating) the K content of the cell maximizes the ability to accurately separate the overlapping K Kß and Ca Kα peaks in the x-ray spectra. For example, rubidium has been effectively substituted for potassium in smooth muscle cells, thus giving an improvement in calcium measurements. Ouabain, a cardiac glycoside widely used in experimental and clinical applications, inhibits Na-K ATPase at the cell membrane and thus alters the cytoplasmic ion (Na,K) content of target cells. In epithelial cells primarily involved in active transport, such as the proximal tubule of the rabbit kidney, ouabain rapidly (t1/2= 2 mins) causes a decrease2 in intracellular K, but does not change intracellular total or free Ca for up to 30 mins. In the present study we have taken advantage of this effect of ouabain to determine the mitochondrial and cytoplasmic Ca content in freeze-dried cryosections of kidney proximal tubule by electron probe x-ray microanalysis.

Electron Probe Analysis and Ultrastructure of Cultured, Freeze Dried Vascular Smooth Muscle

1975 ◽  
Vol 33 ◽  
pp. 558-559 ◽  
Author(s):  
R. E. Garfield ◽  
A. P. Somlyo
Keyword(s):  
Smooth Muscle ◽  
Electron Probe ◽  
Cultured Cells ◽  
Divalent Cations ◽  
Electron Probe Analysis ◽  
X Ray ◽  
Whole Cells ◽  
Probe Analysis ◽  
Freeze Dried ◽  
Ultrathin Sections

Electron probe x-ray microanalysis at a spatial resolution of 1000 Å or better is now feasible and has been used to demonstrate mitochondrial deposits of divalent cations in ultrathin sections of fixed tissues. Monovalent and unbound ions, however, are lost and/or translocated during chemical fixation, necessitating the use of unfixed ultrathin frozen dried sections for electron probe analysis. The use of whole, frozen-dried cultured cells for electron probe analysis is easier than cryo-ultramicrotomy and presents some advantages such as rapid freezing rates attainable and imaging at conventional (75-80 kV) voltages. Smooth muscle cells from guinea pig aortas were cultured on Formvar-coated nylon grids (Fig 1). Grids with attached cells were blotted on filter paper and plunged into liquid nitrogen and dried frozen in a vacuum evaporator.In dried whole cells examined at 75 or 80 kV, the cell outline and processes were distinct and the nuclei and mitochondria containing electron opaque granules identifiable (Fig 2,3).

Materials science of cells: Electron probe x-ray microanalysis and x-ray mapping in biology

1989 ◽  
Vol 47 ◽  
pp. 238-239
Author(s):  
Andrew P. Somlyo
Keyword(s):  
Spatial Resolution ◽  
Electron Probe ◽  
Materials Science ◽  
High Sensitivity ◽  
Specimen Preparation ◽  
X Ray ◽  
Low Concentrations ◽  
Freeze Dried ◽  
And Control

The general aims of Electron Probe X-ray Microanalysis (EPMA) in Biology is similar to that in Materials Science: the determination of composition at sub-micron resolution. Special requirements include stringent precautions for specimen preparation, high sensitivity for detecting low concentrations of elements avoidance, and if not possible, quantitation and control of radiation damage, and spatial resolution of at least tens of nanometers.Special precautions for specimen preparation are dictated by the fact that biological materials exist in an aqueous milieu, and one of the most common objectives of biological EPMA is the localization and quantitation of diffusible elements. Therefore, specimen preparatory techniques must include handling of live tissues in a manner that maintains normal physiological states, and rapid freezing to trap diffusible elements in their physiological compartments. In order to obtain high spatial resolution, ultrathin cryosections have to be obtained, freeze dried and transferred to the microscope under conditions that prevent elemental translocations. Specimen temperatures during cryo-sectioning are usually at about -100°centigrade.

Electron Probe Microanalysis of Zinc-Bearing Hexagonal Ferrites

1965 ◽  
Vol 9 ◽  
pp. 304-313
Author(s):  
J. R. Shappirio
Keyword(s):  
Electron Probe ◽  
Hexagonal Ferrites ◽  
Structural Units ◽  
Electron Probe Analysis ◽  
X Ray ◽  
Layer Structures ◽  
Cell Data ◽  
Unit Cell Data ◽  
The Ideal

AbstractThe electron probe is shown to be an effective tool for the analysis of the series of ferrimagnetic oxides referred to as the hexagonal ferrites. This series of compounds) containing barium, Fe3+, and a divalent metal cation, is formed by an ordered stacking of basic structural units in varying ratios. The ideal, complex stoichiornewy of these polytype-like mixed-layer structures can be computed from X-ray unit cell data; the various structures and their predicted stoichiometry are reviewed. Results of electron probe analysis of zinc-bearing single-crystal hexagonal ferrites are compared with theoretical values, the various correction procedures applied to the probe data are presented, and the limitations of the method in the analysis of hexagonal ferrites are discussed. The information obtained from this study has laid the groundwork for the determination of chemistry in substituted members of the hexagonal ferrite group, and will contribute significantly to the interpretation of the magnetic properties exhibited by these compounds.

scholarly journals Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium.

1982 ◽  
Vol 95 (1) ◽  
pp. 118-126 ◽  
Author(s):  
S D Chung ◽  
N Alavi ◽  
D Livingston ◽  
S Hiller ◽  
M Taub
Keyword(s):  
Epithelial Cells ◽  
Proximal Tubule ◽  
Primary Cultures ◽  
Sugar Transport ◽  
Optimal Growth ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Metabolic Inhibitor ◽  
Gamma Glutamyl Transpeptidase ◽  
Kidney Epithelial Cells

Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone-deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha-methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.

A sample preparation for quantitative determination of magnesium in individual lymphocytes by electron probe X-ray microanalysis

1986 ◽  
Vol 141 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Gregory R. Hook ◽  
Ronald J. Elin ◽  
Jeanette M. Hosseini ◽  
Carol Swyt ◽  
Charles E. Fiori
Keyword(s):  
Sample Preparation ◽  
Quantitative Determination ◽  
Electron Probe ◽  
X Ray

The Determination of the Efficiency of Energy Dispersive X-Ray Spectrometers by a New Reference Material

2006 ◽  
Vol 12 (5) ◽  
pp. 406-415 ◽  
Author(s):  
Marco Alvisi ◽  
Markus Blome ◽  
Michael Griepentrog ◽  
Vasile-Dan Hodoroaba ◽  
Peter Karduck ◽  
...  
Keyword(s):  
Reference Material ◽  
Electron Probe ◽  
Detection Efficiency ◽  
Calibration Procedure ◽  
Energy Dispersive ◽  
X Ray ◽  
Line Intensities ◽  
Operator Assistance ◽  
Scanning Electron

A calibration procedure for the detection efficiency of energy dispersive X-ray spectrometers (EDS) used in combination with scanning electron microscopy (SEM) for standardless electron probe microanalysis (EPMA) is presented. The procedure is based on the comparison of X-ray spectra from a reference material (RM) measured with the EDS to be calibrated and a reference EDS. The RM is certified by the line intensities in the X-ray spectrum recorded with a reference EDS and by its composition. The calibration of the reference EDS is performed using synchrotron radiation at the radiometry laboratory of the Physikalisch-Technische Bundesanstalt. Measurement of RM spectra and comparison of the specified line intensities enables a rapid efficiency calibration on most SEMs. The article reports on studies to prepare such a RM and on EDS calibration and proposes a methodology that could be implemented in current spectrometer software to enable the calibration with a minimum of operator assistance.

Proximal tubule volume regulation in hyperosmotic media: intracellular K+, Na+, and Cl-

1989 ◽  
Vol 257 (6) ◽  
pp. C1093-C1100 ◽  
Author(s):  
L. Rome ◽  
J. Grantham ◽  
V. Savin ◽  
J. Lohr ◽  
C. Lechene
Keyword(s):  
Proximal Tubule ◽  
Cell Volume ◽  
Volume Regulation ◽  
Electron Probe ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Cell Shrinkage ◽  
Volume Increase ◽  
Regulatory Volume Increase ◽  
Expected Increase

Nonperfused proximal S2 segments from rabbit kidney cortex have been shown to keep cell volume constant as medium osmolality is slowly raised but to shrink and not exhibit regulatory volume increase (RVI) if medium osmolality is abruptly elevated (J. Lohr and J. Grantham. J. Clin. Invest. 78: 1165-1172, 1986). In the current study, 0.5 mM butyrate in the medium 1) extended the range from 361 to 450 mosmol/kgH2O over which cells maintained volume constant as osmolality was gradually raised and 2) restored RVI after cell shrinkage when osmolality was rapidly raised from 295 to 400 mosmol/kgH2O. Volume regulation was associated with net increases in intracellular Na+ and Cl- but no change in K+ (measured by electron probe). The increments in Na+ and Cl- were insufficient to account for the total addition of osmolytes required for volume maintenance or restoration. The fraction of the expected increase in intracellular osmoles accounted for by the increase in [(K+)i + (Na+)i + (Cl-)i] was 52 and 21% for gradual and rapid osmotic changes, respectively. We conclude that butyrate enhances the capacity of S2 segments to regulate volume in hyperosmotic medium by promoting addition of Na+ and Cl- and by other undermined factors.

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