Life Sciences Commons^™

Preparation Of Cultured Smooth Muscle Cells From Human Myometrium For X-Ray Microanalysis, Jarin Hongpaisan, Godfried M. Roomans

Scanning Microscopy

Methodological aspects of the use of X-ray microanalysis in physiological and pharmacological experiments on cultured myometrial cells were investigated. Cultured human myometrial cells were grown from biopsies after detaching the fibroblasts. Of the cultured cells, 95-98% showed desmin-like immunoreactivity. Transmission electron microscopy showed that subcultured cells were different from myometrial cells in situ. The effects of washing the cells to remove external salt-rich medium were investigated. All solutions removed the external medium, resulting in lower concentrations of Na and Cl. In the cells washed with 0.3 M mannitol, most of the elemental concentrations were significantly lower than in their …

In Vitro Systems And Cultured Cells As Specimens For X-Ray Microanalysis, Godfried M. Roomans, Jarin Hongpaisan, Zhengzhu Jin, Ann-Christin Mörk, Ailing Zhang

Scanning Microscopy

In vitro systems and cultured cells are recognized as useful systems in many areas of biomedical research, including X-ray microanalysis. To be reliable, in an vitro system should have an elemental composition close to that of the tissue in situ, react in the same way to stimuli, and retain the in situ regulation of ion transport. In the present paper, four of the most commonly used in vitro systems will be reviewed: incubated tissue slices (liver and pancreas), isolated glands (submandibular gland acini, sweat glands), primary cell cultures (sweat glands, endometrium), and cell lines (the colon cancer cell line …

A Yohimbine-Dependent, Uk14,304 Induced Ion Transient In Ht29 Cells Studied By X-Ray Microanalysis, Wei Zhang, Godfried M. Roomans

Scanning Microscopy

The response of HT29 cells to α₂-adrenoceptor agonists was investigated by X-ray microanalysis. Treatment of the cells with 10 μM UK14,304, an α₂-agonist, resulted in a rise of intracellular Cl and Na, suggesting an antisecretory effect. When the cells were exposed to yohimbine or RX821002, both of which are arantagonists, the former had no effect, whereas the latter produced a minor decrease of the cellular Na, Cl and K concentrations. However, when the cells were pretreated with yohimbine, UK14,304 induced a transient change in ion concentrations, which consisted of a rapid increase of intracellular Cl, Na …

Morphological Effects Of Glycosaminoglycans On Calcium Oxalate Monohydrate Crystals, Y. Shirane, Y. Kurokawa, Y. Sumiyoshi, S. Kagawa

Scanning Microscopy

The effects of individual glycosaminoglycan (GAG) species on calcium oxalate monohydrate (COM) crystal growth were studied in vitro by the observation of crystal morphology grown in a supersaturated calcium oxalate solution in the presence of GAGs using optical and scanning electron microscopes. GAGs affected the morphology of COM crystals differently depending on the species. The growth rates of the crystals formed in the presence of chondroitin-6-sulfate (ChS-C) were higher in length and lower in width and thickness than those of control crystals. The incorporation of dermatan sulfate or heparin into the crystals formed in the presence of these GAGs was …

Low Concentrations Of Mercury Induce Changes In Ion Composition Of Cultured Myoblasts, Joanna Wroblewski, Jan Ekstrand, Romuald Wroblewski

Scanning Microscopy

The effects of low concentrations (1 pM, 1 nM, 1 μM) of mercuric chloride on ion distribution in cultured myoblasts were analysed by energy dispersive X-ray microanalysis. An increase in intracellular sodium concentration was observed five minutes after addition of HgC12 to the culture medium. This increase was dose dependent and accompanied by a transient decrease in potassium concentration. Exposure to 1 nM and 1 μM HgC12 led to a two-fold increase in the cytoplasmic chlorine concentration. The higher HgC12 concentration (1 μM) induced morphological alterations in the form of cell membrane blebs, perforations and shrinkage or flattening of the …

Vitamin E Deficiency As A Model Of Precocious Brain Aging: Assessment By X-Ray Microanalysis And Morphometry, Carlo Bertoni-Freddari, Patrizia Fattoretti, Ugo Caselli, Roberta Paoloni, William Meier-Ruge

Scanning Microscopy

Vitamin E (α-tocopherol) is a known biological antioxidant able to quench the lipid peroxidation chain and to protect the cellular structures (e.g., plasma membranes) from the attack of free radicals which are reported to play a primary role in aging. To assess whether the absence of α-tocopherol from the diet of young laboratory animals may be considered a reliable model of precocious brain aging, intracellular ionic content of brain cortex pyramidal cells, ultrastructural features of synaptic contact zones, synaptic mitochondria and perykarial mitochondria positive to the succlillc dehydrogenase (SDH) histochemical reaction with copper ferrocyanide have been investigated by X-ray microanalysis …

Early Elemental And Ionic Changes In Cultured Cells After Stimulation With Epidermal Growth Factor, Julian E. Beesley, Simon R. Hall, Martin J. Page, Karen Affleck

Scanning Microscopy

Stimulation of A43 l cells (a human vulval epidermal cell line) with 50 ng/ml of epidermal growth factor (EGF) in the presence of 1. 7 mM extracellular calcium produced a sharp and sustained rise in intracellular ionic Ca2+, increased elemental Na, decreased K and a rise in Ca. In the absence of extracellular calcium, the initial Ca2+ rise remained but the sustained elevation of intracellular Ca2+ was abolished, Na and K fluxes were variable and the Ca did not change. Increased Na and decreased K was marked at 2 minutes and returned to the control value after 60 minutes. The …

Cryo-Jet Preservation Of Calcium In The Rat Spinal Cord, William B. Greene, Lyle G. Walsh

Scanning Microscopy

In order to determine the distribution of diffusible ions, especially calcium, in rat spinal cord axons before and after trauma, sham, 6 hours post 6 gm/cm and 20 gm/cm trauma cords were cryo-jet frozen in situ and cryo-sections were subjected to electron probe X-ray microanalysis. The results confirm part of the Ca hypothesis, i.e., some spinal cord axons accumulate intracellular Ca after traumatic injury. In addition, 6 hours after trauma, Ca is only accumulated by axons that have also lost homeostasis. Thus, it is likely that observations of the continual rise of Ca in the traumatized spinal cord are due …

Distribution Of Sodium, Magnesium, Chlorine, Calcium, Potassium, Phosphorus And Sulphur In Z-, I- And A-Bands In Mammalian Striated Muscle, Romuald Wróblewski, Lars Edström

Scanning Microscopy

The regularity of the striation of skeletal muscle offers a unique possibility to analyze different bands of the sarcomere in longitudinally cut semithin cryosections. The aim of the present study was to investigate the elemental content of the Z-, I- and A-bands within the sarcomere which may be related to the affinity of an element to different contractile elements and the water content in different bands. The highest potassium levels were found in the Z-band {802 mmol/kg dry weight (d.w.)} as compared to the I-band (697 mmol/kg d.w.) and the A-band (731 mmol/kg d.w.). The difference between A-band and Z-band, …

Data Handling In Quantitative Microanalysis In Biology, V. Starý

Scanning Microscopy

In cell biology, electron probe X-ray microanalysis can reveal the distribution of chemical elements inside a single cell. The full description of a biological system (cell population, tissue) requires a great number of spot measurements. In quantitative analysis, the measurements are subject to experimental errors of several types; moreover, the relations between the resulting values are usually more interesting than the absolute concentrations. Nevertheless, the proper evaluation of quantitative values can discover information more on the object of study.

A system of simple statistical tests is suggested here which can solve several problems. Some concentration values can be far from …

Time-Resolved Cryofixation Methods For The Study Of Dynamic Cellular Events By Electron Microscopy: A Review, Keith P. Ryan, Gerd Knoll

Scanning Microscopy

The preservation of cells for electron microscopy by chemical fixation is a lengthy process, requiring up to 30 minutes for cytoplasmic stabilisation. This time lag enables many changes to occur in specimens so that they may not reflect their living state when they are observed in electron microscopes. Many artefacts can be avoided by using cryofixation, which freezes specimens over a period that is measured in milliseconds, so that specimens are preserved by cryoimmobilisation. This time resolution can be used to study rapid processes in biology and chemistry because, although electron microscopes cannot observe dynamic cellular events directly, processes can …

An X-Ray Microanalytical Study On The Effects Of Ouabain And N-Ethyl Maleimide On The Elemental Concentrations In Malpighian Tubule Cells Of Locusta, Natalia Pivovarova, John H. Anstee, Ken Bowler

Scanning Microscopy

X-ray microanalysis was used to study elemental distribution in Malpighian tubule cells of Locusta migratoria and how these are affected by the replacement of bathing medium K⁺ with Rb⁺ and by inclusion of the transport inhibitors ouabain and n-ethyl maleimide (NEM) in standard (K⁺-containing) and Rb⁺-Ringer (K⁺-free) solutions. Incubation of tubules in standard Ringer containing 1 mM ouabain dramatically affected the intracellular levels of K and Na. The intracellular K concentration fell and Na concentration increased in all regions studied. Despite this, a gradient of increasing K concentration from basal to apical …

A New Versatile System For Freeze-Substitution, Freeze-Drying And Low Temperature Embedding Of Biological Specimens, H. Sitte, L. Edelmann, H. Hässig, H. Kleber, A. Lang

Scanning Microscopy

A universal system for freeze-substitution (FS), freeze-drying (FD) and low temperature embedding (LTE) has been developed, suited to perform standardized procedures of cryoprocessing biological and medical specimens as well as systematic studies of dehydration and embedding at various low and high temperatures. In a 35 I Dewar vessel with 110 mm neck diameter an aluminum tube is mounted to the bottom of the liquid nitrogen (LN_2x) reservoir and extends to the lower part of the cylindrical neck. At its top an aluminum plate serves as a contact surface for either the FS chamber or the FD chamber. FS …

The Preparation Of Cultured Cells For X-Ray Microanalysis, Alice Warley

Scanning Microscopy

X-ray microanalysis of cells in culture is being used increasingly for the study of relationships between element (ion) content and cell function. There is, however, no one single method which can be used for the preparation of all different cell types for study by microanalysis. Cells in suspension are usually concentrated by centrifugation, before cryofixation, cryosectioning, and freeze drying. On the other hand cells grown as monolayers are more often studied as whole cell mounts, which are washed to remove the external medium before cryofixation and freeze drying. The alternative approach, sectioning of cell monolayers is rarely used. Some of …

Light Element X-Ray Microanalysis In Biology, A. T. Marshall

Scanning Microscopy

It is shown that both qualitative and quantitative light element X-ray microanalysis of biological samples is feasible. These analyses were carried out using ultrathin window (UTW) detectors. Quantitative analysis yields a total element analysis with H estimated by difference or "guesstimated". Comparison with calculated concentrations, or concentrations obtained by chemical analysis, shows that X-ray microanalysis of sections, by the peak to continuum ratio model, give sufficiently accurate results for biological purposes.

The measurement of O concentrations to yield water content is carried out using x-ray imaging techniques, so that the distribution of heavier elements can be spatially related to water …

Optimal Freeze-Drying Of Cryosections And Bulk Specimens For X-Ray Microanalysis, L. Edelmann

Scanning Microscopy

Electron microscopic investigations of rapidly frozen specimens of striated muscle, either frozen-hydrated or obtained after different dehydration procedures, have shown that the subcellular distribution of the main cellular cation K⁺ or its surrogates Rb⁺, Cs⁺, or Tl⁺ does not follow the water distribution but follows certain proteins. Conflicting results obtained by X-ray microanalysis of freeze-dried cryosections are explained by showing that freeze-drying of bulk specimens and cryosections must be carried out for rather long periods at low temperature in order to avoid severe shrinkage and ion redistribution artefacts. Proposals for future freeze-drying studies are …

Electron Probe X-Ray Microanalysis Of Epithelial Cells: Aspects Of Cryofixation, Karl Zierold, Hartmut Hentschel, Frank Wehner, Armin Wessing

Scanning Microscopy

Content and distribution of diffusible ions in epithelial cells were studied by scanning transmission electron microscopy and energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections from trout kidney, rat liver and Malpighian tubules of Drosophila larvae. Cryofixation of small excised kidney and liver samples by rapid immersion into liquid propane resulted in intracellular K/Na-ratios < 1. In contrast, K/Na-ratios > 7 were obtained after in situ cryofixation by means of a cryopunching device which allows tissue pieces to be frozen during excision from the intact organ. Isolated hepatocytes cryofixed in a small droplet of culture medium had a K/Na-ratio of 3.7. After culturing the hepatocytes, …

Changes Of Ion And Water Content Of Mouse Intestinal Cells After Pilocarpine And Isoproterenol Stimulation, Thomas Von Zglinicki, Godfried M. Roomans

Scanning Microscopy

Cytoplasmic monovalent ion and water contents in morphologically defined mice jejuna) cells were measured by X-ray microanalysis in order to gain insight into the cell-type specificity of intestinal electrolyte transport mechanisms. Ion and water contents were measured independently. It was found that in some cases net fluxes of ions and water do not correspond to the assumption of constant osmotic activity of cytoplasmic Na and K ions. Stimulation of secretion with the cholinergic secretagogue pilocarpine resulted in efflux of Cl^- from and influx of K⁺ into crypt enterocytes containing small secretion granula (crypt A cells). No significant changes …

X-Ray Microanalysis With The Environmental Scanning Electron Microscope: Interpretation Of Data Obtained Under Different Atmospheric Conditions, D. C. Sigee, C. Gilpin

Scanning Microscopy

X-ray microanalysis of non-biological and biological specimens was carried out in an environmental scanning electron microscope (ESEM) over a range of atmospheric conditions. Introduction of water vapour into the specimen chamber lead to direct X-ray contribution from oxygen atoms, an increase in extraneous background (causing reduced P/B ratios of other elements), X-ray absorption (also reducing P/B ratios) and broadening (skirting) of the electron beam. Similar results were obtained after introduction of an argon atmosphere. These effects were reduced under conditions of minimal chamber atmospheric pressure and maximal accelerating voltage.

Because of beam skirting, quantitative X-ray microanalysis of biological specimens in …

Use Of In Vitro Systems For X-Ray Microanalysis, Jarin Hongpaisan, Ann-Christin Mörk, Godfried M. Roomans

Scanning Microscopy

The use of X-ray microanalysis in human pathology may require the use of cryoprepared tissue. Often it is impossible to carry out freezing of the tissue in an optimal way, and in addition, it is difficult to carry out experiments in living patients. The use of in vitro systems and cell cultures allows separation of the process of tissue removal and the freezing procedure, and also makes testing of pharmacological or toxic substances possible. In experiments with animal tissue it was shown that incubation in a physiological buffer induced significant changes in the concentrations of Na, K, and Cl. In …

Standardless Analysis Of Biological Tissue Sections, W. A. P. Nicholson

Scanning Microscopy

The X-ray microanalysis of thin biological samples which are usually supported on a thin organic film or are self-supporting specimens, has required the use of standards which contain the elements of interest. Spectra from the standards are used to calculate the factors for converting X-ray data recorded on the specimen into elemental concentrations. A method is discussed here, in which these factors are evaluated from formulae. The most important physical process to be evaluated is that of characteristic X-ray production in the specimen. The bremsstrahlung production must also be evaluated if the Hall or continuum normalisation (CN) method of quantitation …

X-Ray Microanalysis Of Camp-Induced Ion Transport In Ncl-Sg3 Sweat Gland Cells, Ann-Christin Mörk, Godfried M. Roomans

Scanning Microscopy

cyclic AMP (cAMP)-induced ion transport in a human sweat gland cell line, NCL-SG3, was investigated by X-ray microanalysis. Stimulation with cAMP caused a decrease in cellular Cl and K content in cells grown on impermeable as well as permeable supports. In cells grown on plastic coverslips or Transwell inserts stimulation with cAMP caused a significant decrease of the Na concentration, but no significant effect on the intracellular Na was seen on cells grown on grids. cAMP had no significant effect on the intracellular Ca concentration.

Analysis Of Heavy Metals In Mouse Uterine Secretion By X-Ray Microanalysis, Zhengzhu Jin, B. Ove Nilsson

Scanning Microscopy

A method for the detection of heavy metal ions in the uterine secretion of the mouse was tested. Mice received an intravenous injection of lead, and beads of an analytical grade resin, Chelex 100, were deposited in the uterine lumen to absorb heavy metals present in the secretion. X-ray microanalysis of the flushed beads showed that lead was present in a dose-related way in the uterine secretion of mice that had received 0.05, 0.1, or 0.15 mg lead per g body weight. Thus, the technique allows quantitative analysis of lead in the uterine secretion. The amount of lead present in …

Distribution And Intracellular Localization Of Titanium In Plants After Titanium Treatment, G. Kelemen, A. Keresztes, E. Bacsy, M. Feher, P. Fodor, I. Pais

Food Structure

The physiological role of titanium in plants has not been elucidated yet, but a higher rate of growth , greater chlorophyll content and higher productivity, among others , may be attributed to this element. We have investigated the distribution of titanium in wheat seedlings after Titavit (a Ti ·ascorbate containing plant conditioner) treatment applied either through the leaf or through the root. In field experiments, we also sprayed spinach plants with Titavit. We have found a practically unidirectional tran slocation of Ti from the shoot into the root, and a preferential local ization of Ti in the nuclear cell fraction …

A Tutorial And Comprehensive Bibliography On The Identification Of Foreign Bodies Found In Food, D. F. Lewis

Food Structure

The paper presents a philosophical approach to the investigation of foreign body incidents in food. The suggested procedure has similarities with "key" procedures used for taxonomy. However, the wide diversity of materials encountered and the need to determine something of the history of the offending item demands modifications to this process. The categorisation may be made on instinct or experience, and appropriate confirmatory tests will be applied in the light of the initial categorisation. Cardinal rules are proposed: firstly , the receipt of the items should be well documented; and secondly, the initial observations should be as non-destructive as possible. …

Electron Probe X-Ray Microanalysis Applied To Thin Surface Films And Stratified Specimens, J. L. Pouchou, F. Pichoir

Scanning Microscopy

The basic principles of X-ray microanalysis of thin surface films and stratified targets are summarized. The influence of the electron beam accelerating voltage on the analyzed depth is discussed. It is shown that in the field of soft X-rays, the effective depth of analysis is most often limited by the absorption of the radiation in the specimen itself. For high energy radiations, the importance of the secondary emission due to fluorescence excited by the continuous radiation and by the characteristic lines is outlined. The main concepts of the recent ø(ρz) models used for quantitation in the recent software packages are …

X-Ray Microanalysis Of Epithelial And Secretory Cells In Culture, Anne Von Euler, Eva Pålsgård, Christina Vult Von Steyern, Godfried M. Roomans

Scanning Microscopy

Cell cultures can be used to study ion transport processes. X-ray microanalysis of cell cultures at the cellular level gives interesting information that can complement electrophysiological and tracer studies. In this paper, methods for culturing and preparing a variety of epithelial and secretory cells (fibroblasts, insulinoma cells, bovine mammary epithelial cells, colon cancer cells) for X-ray microanalysis are presented. Results show that sometimes cell cultures are not homogeneous with respect to ion content or reaction to physiological stimuli. In colon cancer cell cultures, a "high K" and a "low K" cell subpopulation was found; these subpopulations also differed with respect …

Microanalysis Of Individual Silver Halide Microcrystals, Shijian Wu, A. Van Daele, W. Jacob, R. Gijbels, A. Verbeeck, R. De Keyzer

Scanning Microscopy

Elemental distributions and contents of tabular and cubic silver halide microcrystals were analyzed by backscattered electron imaging, scanning transmission electron imaging, X-ray mapping and X-ray analysis in the spot mode by scanning transmission electron microscopy (STEM) combined with energy-dispersive X-ray spectrometry (EDXS). By using a liquid nitrogen cryostage, damage to the microcrystals and drift of the sample under electron bombardment were minimized. Monte Carlo simulation shows the electron trajectories in the silver halide microcrystals for different experimental conditions and directs the selection of the beam position for X-ray spot analysis. The backscattered electron images, scanning transmission electron images and X-ray …

Effect Of Chronic Treatment With Diuretics On Mouse Liver: A Morphological And Microanalytical Investigation, Ann-Christin Mork, Anne Von Euler, Godfried M. Roomans

Scanning Microscopy

In an attempt to produce an animal model for the disease cystic fibrosis (CF), mice were treated chronically with the diuretics amiloride and furosemide, in order to cause chronic inhibition of transepithelial ion transport. Experiments were carried out on adult mice (2 months treatment); in addition, pregnant mice were treated with diuretics, and tissue from offspring 2 and 7 days post partum was investigated. Since biliary cirrhosis is a common occurrence in CF, hepatocytes in the treated mice were investigated by X-ray microanalysis and by light and electron microscopy.

Treatment with amiloride caused a significant decrease in cellular Na concentration …

Comparison Of Cryopreparation Techniques For Electron Probe Microanalysis Of Cells As Exemplified By Human Erythrocytes, Karl Zierold

Scanning Microscopy

Erythrocytes in human blood were used to evaluate the reliability of cryopreparation techniques for electron probe X-ray microanalysis of biological cells and tissues. The elemental content determined by X-ray microanalysis of ultrathin freeze-dried cryosections was found to be consistent with data known from the literature. Considerable redistribution of the intracellular elemental composition was found after freeze-substitution as well as after freeze-drying followed by resin embedding. Two conclusions are drawn from this study: 1. Erythrocytes in human blood are a suitable reference specimen for evaluation of specimen preparation techniques for microanalysis. 2. At present, freeze-dried cryosections are the most reliable specimen …