Life Sciences Commons^™

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 …