Bulk Specimen Preparation For X-Ray Microanalysis of Plant Cells

Author(s):  
Gilbert G. Ahlstrand ◽  
Richard J. Zeyen

Numerous plant pathological investigations have been published where pathologists determined elemental differences between diseased and healthy plant tissue using unfractured bulk specimens and energy dispersive x-ray microanalysis (EDX). In these studies effects of specimen preparation procedures were largely ignored (1). Our objective was to compare bulk specimen preparation procedures using healthy leaf epidermal (surface) cells of barley, Hordeum vulgare, (Fig. 1) to determine advantages and disadvantages of each procedure for reference to future pathological work using EDX. Three preparation procedures were compared: 1) Frozen-hydrated (FH) specimens to maintain soluble and insoluble elements in situ; 2) Freeze-dried (FD) specimens to maintain total soluble and insoluble elements and allow deeper beam penetration than does FH, and; 3) Formalin/acetic acid/ethanol (FAA) fixed specimens, dehydrated in ethanol, and critical point dried (CD) using CO2, for determination of elemental loss in liquid fixatives (2,3).Aluminum specimen stubs were colloidal graphite coated (leaf segments attached to graphite areas) leaving clean Al margins for calibration.

Author(s):  
Andrew P. Somlyo

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.


Author(s):  
A. LeFurgey ◽  
P. Ingram ◽  
L.J. Mandel

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.


2020 ◽  
Author(s):  
Keishiro Yamashita ◽  
Kazuki Komatsu ◽  
Hiroyuki Kagi

An crystal-growth technique for single crystal x-ray structure analysis of high-pressure forms of hydrogen-bonded crystals is proposed. We used alcohol mixture (methanol: ethanol = 4:1 in volumetric ratio), which is a widely used pressure transmitting medium, inhibiting the nucleation and growth of unwanted crystals. In this paper, two kinds of single crystals which have not been obtained using a conventional experimental technique were obtained using this technique: ice VI at 1.99 GPa and MgCl<sub>2</sub>·7H<sub>2</sub>O at 2.50 GPa at room temperature. Here we first report the crystal structure of MgCl2·7H2O. This technique simultaneously meets the requirement of hydrostaticity for high-pressure experiments and has feasibility for further in-situ measurements.


2005 ◽  
Vol 38 (4) ◽  
pp. 678-684 ◽  
Author(s):  
Balder Ortner

A method for the X-ray determination of lattice-plane distances is given. Similar to Bond's method, it is based on the measurement of rocking curves, with some advantages and disadvantages compared with the former method. The new method is especially designed for single-crystal stress measurement. Its usefulness is demonstrated in two examples of lattice-constant and stress measurement.


2020 ◽  
Vol 13 ◽  
pp. 247
Author(s):  
N. Evaggeliou ◽  
Ch. Lykomitrou ◽  
A. Zafiropoulou

In the present study a comparative evaluation of two methods for 137Cs determination (pretreatment for gamma spectrometry) is attempted. One of them is the conventional AMP (ammonium molybdophosphate, (ΝΗ4)3Ρ(Μo3O10)4) method (radiochemical treatment based on coprecipitation) and the other one is a method based on pre-concentration of cesium in situ by using the Mark III Centrifugal Pump. The pump, which is described analytically in the study, is composed of a mo­tor (pump), four cartridge housings (containing the scavengers), a flow meter and a pressure tube (containing the battery pack and the timer board). For justification, this method is compared with the AMP co-precipitation one. Following up the radioanalytical procedures, the gamma spectrometry system (relative efficiency of high purity germanium detector 90%) is also demonstrated, as conformed to mea­ suring obtained parameters. Finally, the advantages and disadvantages of these two methods are recorded and the application of each one is suggested.


2010 ◽  
Vol 66 (6) ◽  
pp. 579-584 ◽  
Author(s):  
Kevin Robertson ◽  
David Bish

X-ray powder diffraction (XRD) data were used to solve the crystal structures of phases in the magnesium perchlorate hydrate system, Mg(ClO4)2·nH2O (n = 4, 2). A heating stage and humidity generator interfaced to an environmental cell enabled in-situ XRD analyses of dehydration reactions under controlled temperatures and partial pressures of H2O (P_{{\rm H}_2{\rm O}}). The crystal structures were determined using an ab initio charge-flipping method and were refined using fundamental-parameter Rietveld methods. Dehydration of magnesium perchlorate hexahydrate to tetrahydrate (348 K) results in a decrease in symmetry (space group = C2), where isolated Mg2+ cations are equatorially coordinated by four H2O molecules with two [ClO4]− tetrahedra at the apices. Further dehydration to the dihydrate (423 K) leads to bridging of the isolated packets to form double corner-sharing chains of octahedra and polyhedra (space group = C2/m).


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