The Hall Method in the Quantitative X-Ray Microanalysis of Biological Specimens: A Review

In the two decades since its inception by T.A. Hall, the continuum theory of quantification has become the general method for quantitative analysis of biological specimens. Although the method was originally developed for thin specimens, its use has been extended to thicker specimens, and it has also been used in quantitative determinations of local water content. The single most important difficulty in the application of the Hall method is the accurate calculation of the extraneous continuum, i.e., the continuum due to non-specimen sources. The different variations in methods for quantitative analysis of local water content are critically compared and a generally applicable method is proposed

Cryopreparation of Tissue for Clinical Applications of X-Ray Microanalysis

A number of diseases is associated with changes in ion and/or water distribution at the tissue or cell level, and X-ray microanalysis can be used to investigate the relationship between the disease process and the changes in elemental distribution. For analysis of diffusible elements by X-ray microanalysis, the tissue has to be prepared by cryotechniques. To carry out studies of this kind in a clinical environment poses a number of problems. Some of these problems occur already before the tissue is frozen, others are caused by the practical and ethical limitations that are imposed on the freezing method itself when human tissue is to be used. The use of cryostat sections for analysis at the cellular level, and of in vitro systems and cell cultures in which sampling and cryopreparation can be separated in time and place can be useful alternatives

The Correction for Extraneous Background in Quantitative X-Ray Microanalysis of Biological Thin Sections: Some Practical Aspects

The correction for extraneous continuum is of great importance in the quantitative analysis of thin sections of biological tissue. Although a theoretical model for this correction is available, its application in practice meets with problems. In this paper, a model system, consisting of sections of homogeneous plastic on copper mesh grids was used to identify sources of inaccuracies in the quantitative procedures. An unmodified electron microscope was operated under standard analytical conditions. It appeared that geometrical factors connected with the position of the analysis relative to the grid bars were of prime importance. The correction for the contribution of the support film to the continuum should ideally be carried out at the same location with respect to the grid bars as the matching measurement on the section. Also the position of the analysis with respect to its coordinates on the grids is important, in particular when the possibility of absorption of X-rays by the specimen holder exists. The use of slot grids (rather than mesh grids) may alleviate this problem at least in part. Additional factors of importance are differential mass loss in specimen and film, as well as undetected variations in specimen current

Calcium and Cystic Fibrosis

Cystic fibrosis (CF) is a generally lethal, congenital, genetic disease of unknown etiology. It is likely that a defective regulation of ion and water transport in exocrine glands and possibly also in other epithelial cells has a central role in the pathogenesis of this disease. Calcium has been implicated in the basic defect underlying CF because of findings of abnormally high calcium levels in some secreted fluids and some cells of CF patients. Using X-ray microanalysis, we have demonstrated elevated calcium concentrations in cultured fibroblasts and in goblet cells of the bronchial epithelium of CF patients. A factor produced by CF fibroblasts in culture can increase the calcium concentration in healthy cells, although this may be an indirect effect. In animal models for CF, such as the chronically reserpinized rat and the chronically isoprotere-nol-treated rat, abnormally high calcium levels in the acinar cells of the submandibular gland could be demonstrated, similar to the situation in CF patients. In the acinar cells of the parotid gland in these animal models, the calcium levels are, however, abnormally low. This suggests that the changes in cell calcium content are secondary to other changes, possibly changes in the secretory proteins. A study of the effect of the serum calcium level and of the calciotropic hormone calcitonin suggested that neither of these factors could be directly linked with CF. It is concluded that several lines of evidence point to a secondary rather than a primary role for calcium in the pathogenesis of CF

A Yohimbine-Dependent, UK14,304 Induced Ion Transient in HT29 Cells Studied by X-Ray Microanalysis

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 and a decrease of K at 1 and 5 minutes. Concurrently, a marked increase in intracellular Ca was found. This transient change could also be induced by different doses of UK14,304 (100 nM or 10 μM) after preincubation with yohimbine (0.3 μM or 3 μM). In contrast, UK14,304 did not elicit a similar response after pretreatment of the HT29 cells with RX821002. Also, yohimbine did not affect the ion content of the cells after pretreatment with UK14,304. These results indicate that yohimbine may have a specific effect by which the cells are sensitized to the ion transient induced by UK14,304

Post-Mortem Storage of Tissue for X-Ray Microanalysis in Pathology

Possible alternatives to rapid freezing in liquid nitrogen of tissue for X- ray microanalysis of electrolytes at the cellular level were investigated. These alternatives might be used in cases where tissue becomes available for examination, e.g., at autopsy, but liquid nitrogen is not immediately available. Rat submandibular gland was used as a test tissue. Freezing of pieces of tissue in a conventional freezer at -80°C or even at -20°C retained the elemental distribution at the cellular level, and also retained the difference between a \u27normal\u27 and a \u27pathological\u27 (mimicked by an inject ion of a high dose of isoproterenol) situation. Storage of tissue in a refrigerator, or delaying the autopsy in anticipation of the arrival of liquid nitrogen is not recommended. Significant changes in the cellular ion content occurred if the tissue was left in the animal for 24h post-mortem

Intracellular Localization of Heavy Metals in Yeast by X-Ray Microanalysis

The intracellular localization of heavy metals in yeast cells was studied by means of energy-dispersive X-ray microanalysis. The yeast, Saccharomyces cerevisiae, was pretreated with phosphate and then loaded with different metal ions, by suspending the cells in salt solutions (Ni, Zn, Cd, Pb, Al and Cr). For the analysis, the cells were embedded in gelatin, rapidly frozen, and thin cryosections were cut on a dry knife. A considerable uptake of divalent cations by the yeast cells was found to occur. The cations were bound to the polyphosphate granules localized in or close to the cell vacuoles. Immediately after phosphate loading, the polyphosphate granules were predominantly located in the cytoplasm, but as the incubation progressed, they migrated to the vacuole. As for trivalent cations, Cr was taken up and also stored in the polyphosphate granules, but Al could not be demonstrated with certainty in the cells, only in the cell walls. Incubation of the cells with zinc, cadmium or lead ions caused a significant decrease of the relative size of the vacuole

Preparation of Cultured Smooth Muscle Cells from Human Myometrium for X-Ray Microanalysis

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 unwashed counterparts and those washed in the other solutions. In cells washed in either 0.15 M ammonium acetate or distilled water, no significant differences in P and K compared with their unwashed counterparts were found. There were also no significant differences between cells washed in ammonium acetate and in distilled water. In subsequent experiments ammonium acetate was used. Incubation of cells in standard Ringer\u27s solution resulted in an increase in Na and Cl, and a decrease in K, concomitantly with an increase in Ca. Although Ringer\u27s solution per se can elicit changes in diffusible elements in the cells, physiological and pharmacological effects of oxytocin could still be detected in Ringer\u27s solution. However, effects of oxytocin were different when the experiment was done in culture medium, instead of in Ringer\u27s solution

Element Concentrations in the Intestinal Mucosa of the Mouse as Measured by X-Ray Microanalysis

Subcellular ion distribution in villus, crypt, Paneth and smooth muscle cells of the mouse small intestine under resting conditions was investigated by X-ray microanalysis of ultrathin cryosections. In addition, the mass distribution was estimated by measuring the optical transmission of the compartments in transmission electron micrographs. Each cell type is characterized by a special composition in terms of the major monovalent ions Na, K, and Cl. In particular, among crypt epithelial cells, those cells containing small secretion granula (termed crypt A cells) also display cytoplasmic ion concentrations significantly different from crypt epithelial cells lacking secretion granula (crypt B cells). Monovalent ion concentrations in the cytoplasm of Paneth cells, muscle cells, and crypt epithelial cells lacking secretion granula are higher than expected from osmotic considerations. Hence, significant binding of ions to cytoplasmic polyelectrolytes is assumed in these cells. There are gradients of dry mass and K concentration from the luminal to the basal side of the cell, both in crypt and in villus cells. The terminal web in these cells is rich in Na and Cl. The elemental composition of the large, dark secretion granula in Paneth cells is similar to that of the small dark granula in crypt cells. However, the two morphologically different types of granula within the Paneth cells have a significantly different elemental composition, which might reflect maturation of secretion granula

Changes of Ion and Water Content of Mouse Intestinal Cells After Pilocarpine and Isoproterenol Stimulation

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 in ion concentrations were found in crypt enterocytes without secretion granula (crypt B cells). Crypt A cells were more likely to be stimulated by pilocarpine than crypt B cells, and the basolateral K+ efflux pathway in crypt A cells appeared to be rate-limiting. In villus enterocytes, pilocarpine stimulated Cl- efflux. Isoproterenol caused marked changes in the cytoplasmic Cl content of all epithelial cells. These changes were reversed by inhibition of adenylate cyclase by alloxan, with the sole exception of Cl increase in villus absorptive cells. The results are consistent with an cAMP-mediated stimulated secretion in crypt epithelial cells and a predominantly cAMP-independent stimulation of absorption in villus cells. The results obtained suggest a transcellular route of Cl absorption in the mouse jejunum