The application of energy dispersive X-ray microanalysis in studies of irritant contact reactions. A mini-review.
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Electron Probe Microanalysis↗
Biomedical subjects
G M Roomans
Publications and source records attributed to G M Roomans.
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.
Copper↗
Elemental microanalysis of biological specimens.
Although X-ray microanalysis in the electron microscope is the most common method for microanalysis of biological specimens, other methods of elemental microanalysis (electron energy loss spectroscopy, scanning Auger microanalysis, and proton, ion, and laser microprobe analysis) may provide important complementary information and help overcome some of the limitations of electron probe X-ray microanalysis. Despite differences in physical principles and instrumentation, the various microanalytical methods have much in common with regard to specimen preparation, quantitative analysis, and interpretation of analytical data. A common approach to microanalytical problems in the biological sciences, irrespective of the analytical techniques used, seems therefore indicated.
Electron Probe Microanalysis↗
Low temperature techniques in biomedical microanalysis.
Many diseases are associated with a change in the distribution of diffusible ions at the cell or tissue level. These diseases can profitably be studied by X-ray microanalysis. This technique for the study of ion distribution requires the use of cryoprepared specimens. Analysis at low or medium resolution can be carried out on thick or semi-thick cryosections, or on frozen-hydrated or freeze-dried embedded bulk samples. Such analyses are particularly useful in the initial stages of an investigation, or when many data from a large number of samples have to be acquired. Quantitative analysis is then usually carried out with the peak-to-local background method: by taking the background in the same energy range as the characteristic peak, an intrinsic correction for absorption of X-rays within the sample is made. X-ray microanalysis of cultured cells prepared by freeze-drying can also be carried out in this way. Analysis at high resolution has to be carried out on thin sections: cryosections, or sections of freeze-substituted or freeze-dried embedded tissue. For the latter type of specimens, the use of low-temperature embedding methods may have important advantages. Quantitative analysis of thin sections is carried out by the established continuum method.
Animals↗
Early effects of alloxan on rat submandibular gland.
Early effects of alloxan on structure and function of rat submandibular gland were investigated by light and electron microscopy, X-ray microanalysis, and by analysis of secreted saliva. Alloxan induced, already within 3 h, intracellular accumulation of mucus, as also evidenced by an increase in the cellular calcium concentration. In addition, 3 to 6 h after injection of alloxan, the granular duct cells were found to be vacuolated, which could suggest a cytotoxic effect of alloxan on this cell type. Alloxan did not significantly affect the flow rate of submandibular saliva after pilocarpine stimulation, and also did not significantly alter the concentrations of sodium, potassium, calcium and protein in the final saliva. The effects of alloxan on the acinar cells of the rat submandibular gland and the absence of effects on fluid secretion by this gland are in accordance with a direct or indirect inhibition of the cAMP system.
Alloxan↗
Effects of reserpine treatment on the ultrastructure of rat parotid and submandibular gland.
The chronically reserpine-treated rat has been used as an animal model for the disease cystic fibrosis, a generalized exocrinopathy. Ultrastructural changes in the parotid and submandibular gland occurring during the seven-day treatment with reserpine were studied by transmission electron microscopy. In the parotid gland, marked changes were observed in the size and structure of the zymogen granules. In fasted control animals, most parotid acinar cells have granules with a virtually homogeneous, electron-dense appearance. During the first three days of treatment, most cells have enlarged, electron-translucent granules. After the third day, and most pronounced at day six and seven, many granules are irregularly electron-dense. In the submandibular gland, reserpine causes a gradual increase in the relative volume of the cell that is occupied by mucus. The ultrastructural changes are discussed in relation to metabolic and biochemical effects of reserpine treatment on salivary glands.
Animals↗
The human sperm nucleus takes up zinc at ejaculation.
Ejaculated and vasal sperm were obtained from men referred for vasectomy, and sperm nuclear elements were determined by X-ray microanalysis. Sperm head zinc concentrations, expressed as the ratio Zinc to Sulphur, were significantly higher in ejaculated than in vasal sperm. A physiological sperm nuclear zinc uptake is discussed in relation to sperm chromatin decondensation.
Adult↗
Saliva and sweat secretion in man during a one-hour calcium clamp.
The effect of a 1-h calcium infusion, directly regulated to obtain and maintain a predetermined blood calcium level ('calcium clamp'), on the composition of parotid saliva, mixed saliva, and sweat was determined in healthy volunteers. An increase in amylase content of the parotid saliva under hypercalcaemic conditions was observed, but the 'calcium clamp' had no effect on flow rate, total and ionized calcium, sodium, potassium, and phosphate levels as well as pH in parotid and mixed saliva. The sweat calcium concentration was negatively correlated with sweat rate, in contrast to sodium and chloride concentrations. Increasing serum calcium levels did not affect ionic composition of the sweat and sweat flow rate.
Adult↗
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 isoproterenol-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.
Animals↗
X-ray microanalysis of goblet cells in bronchial epithelium of patients with cystic fibrosis.
The elemental composition of goblet cells in bronchial epithelium was investigated by X-ray microanalysis. Biopsies were obtained from the left main bronchus, shock-frozen and freeze-substituted. In comparison to the control group (patients with chronic bronchitis), the goblet cells of patients with cystic fibrosis (CF) contained significantly more calcium. The analysis also indicated higher sulphur levels and lower potassium levels in the goblet cells of CF patients. Detailed analysis of a control biopsy showed that the calcium level in the mucous cells of the submucosal glands was considerably higher than that in the goblet cells. Although the data point to a higher calcium content of goblet cell mucus in CF patients, the significance of this finding for the calcium content of the tracheobronchial secretions in CF patients is unclear.
Adult↗
Quantitative correlative proton and electron microprobe analysis of biological specimens.
To investigate the possibility of quantitative correlative proton microprobe (PMP) and electron microprobe (EMP) analysis of biological soft tissue, model specimens were analyzed by both techniques. The specimens consisted of freeze-dried sections of gelatin containing known concentrations of nickel chloride. Both for PMP and for EMP, the signal was expressed as the ratio of the characteristic intensity and the continuum intensity in a peak-free region of the spectrum. With both techniques, calibration curves (signal versus known concentration) obtained, showed a deviation from linearity at high nickel concentrations. However, a linear relation (correlation coefficient 0.996) was obtained in a plot of EMP signal versus PMP signal. This indicates that quantitative correlative PMP and EMP analysis can be carried out by using the same standard for both analytical techniques.
Animals↗
The chronically reserpinized rat: decreased glycolytic activity in the submandibular gland.
Some of the enzymes and metabolites of the glycolytic pathway of an animal model for cystic fibrosis (the chronically reserpine-treated rat) were investigated. The activities of the enzymes phosphofructokinase (P less than 0.002), enolase (P less than 0.03), pyruvate kinase (P less than 0.005), and lactate dehydrogenase (P less than 0.009) were decreased whereas the activity of glycerol-3-phosphate dehydrogenase was unaffected in the submandibular glands of the treated animals. For metabolites, the reserpine treatment resulted in an increased concentration of glycogen (P less than 0.0002) and phosphoenolpyruvate (P less than 0.001) and a decreased concentration of pyruvate (P less than 0.005) and lactate (P less than 0.002) in the glands. The concentration of glucose and glycerate-2-phosphate was unaffected. The perchloric acid-soluble part of the proteins was also increased (P less than 0.0001) in the submandibular glands of the reserpine-treated animals, as was the activity of ribonuclease. These findings point to a disturbance in the metabolism of glucose and a possible acidosis in the submandibular glands of this animal model for cystic fibrosis.
Animals↗
The chronically pilocarpine-treated rat in the study of cystic fibrosis: investigations on submandibular gland and pancreas.
The chronically pilocarpine-treated rat has been proposed as an animal model for the disease cystic fibrosis, a generalized exocrinopathy. The effect of chronic pilocarpine treatment on structure, composition, and function of the acinar cells of rat submandibular gland and pancreas was investigated by electron microscopy, X-ray microanalysis, and biochemical analysis. The morphological effects of chronic pilocarpine treatment were most pronounced in the pancreas. The number and size of the zymogen granules was increased, and the granules had a less electron-dense appearance. X-ray microanalysis at the cellular level showed in both the submandibular gland and the pancreas a significant increase in calcium and a decrease in sodium. The increase in cellular calcium concentrations can be explained by an increase in the relative volume of secretory material in the cell (assessed by morphometry) and an increase in the local calcium concentration in the secretory material (assessed by X-ray microanalysis at the subcellular level). Chronic pilocarpine treatment caused a disturbance of glycolysis and energy metabolism in the submandibular gland, but no significant effects in this respect were noted in the pancreas. On the other hand, a nearly twofold increase of the pancreatic amylase activity was noted. The pancreas appeared somewhat hyperreactive towards cholinergic stimulation.
Animals↗
Effect of chronic treatment with cystic fibrosis fibroblast medium on rat submandibular gland acinar cells.
The effect of chronic treatment with cystic fibrosis (CF) fibroblast medium on rat submandibular gland and pancreas was investigated. Rats were injected for 8 days with conditioned medium from normal or CF fibroblasts. The elemental content of the acinar cells was measured by X-ray microanalysis of cryosections. A significant increase in cellular calcium, and a decrease in cellular sodium concentrations were found after treatment with CF medium. The ultrastructure of the submandibular acinar cells was not affected by the conditioned CF fibroblast culture medium. No effect of treatment with CF medium on ultrastructure and elemental content of pancreatic acinar cells could be demonstrated. The response to alpha-adrenergic, beta-adrenergic, cholinergic, and peptidergic stimulation in submandibular gland acinar cells of rats injected with normal or CF medium was investigated in vitro. With regard to changes in elemental composition after stimulation, no significant differences in response between the two groups could be found. Apparently, a factor in conditioned medium from cultured CF fibroblasts induces a net increase in calcium content of rat submandibular gland acinar cells. Possibly, this factor acts in a similar way in CF patients and may cause elevated calcium levels in CF cells.
Adolescent↗
X-ray microanalysis of psoriatic skin.
Electron probe x-ray microanalysis was used to study elemental distribution in uninvolved and involved skin from patients with psoriasis, and in skin from healthy controls. Significant differences were found between the involved and uninvolved psoriatic skin. In the involved skin, the concentrations of Mg, P, and K were higher in the stratum germinativum, spinosum, and granulosum, compared to the corresponding strata in uninvolved skin. Neither involved nor uninvolved psoriatic stratum germinativum differed markedly from nonpsoriatic control stratum germinativum. In uninvolved psoriatic skin only a lower level of K was noted. In comparison to uninvolved psoriatic skin, the elemental composition of the various strata of involved psoriatic skin shows a pattern typical for highly proliferative, nonneoplastic cells.
Adult↗
Effects of reserpine and isoproterenol on elemental distribution in submandibular gland of rat.
The effects of a single dose of reserpine or isoproterenol on the acinar cells of rat submandibular gland were investigated by electron microscopy and X-ray microanalysis. About 4 h after injection of reserpine, the cellular Ca concentration started to increase and at 24 h a level two to three times the control value was reached. The mucus content of the cells increased in parallel with the Ca concentrations. Isoproterenol stimulation depleted the acinar cells of their mucus. Resynthesis of mucus was visible at 8-12 h after injection. Changes in cellular Ca concentrations followed the mucus content of the cells. After 24 h both the mucus content and the Ca concentration were significantly increased over the control values. High-resolution X-ray microanalysis on thin cryosections showed that the calcium was accumulated in the mucus granules, both after reserpine and after isoproterenol administration. The newly synthesized mucus was also more calcium-rich than the original mucus. The effect of a single isoproterenol stimulation on elemental content of rat submandibular gland acinar cells was not significantly different from that of chronic isoproterenol treatment. Chronic treatment with reserpine resulted in a higher cellular Na/K ratio than a single dose.
Animals↗
X-ray microanalysis of resting and stimulated rat pancreas.
The elemental distribution in acinar cells of rat pancreas was investigated by X-ray microanalysis of thin, freeze-dried cryosections. In the resting cell, the highest calcium concentrations were found in the basal part of the cell (including the endoplasmic reticulum) and in the zymogen granules. Mitochondrial calcium concentrations were low. Zymogen granules were rich in sulphur, but low in phosphorus, sodium and potassium. Stimulation of the pancreas by perfusion in vivo with the cholinergic agonist carbachol caused a significant decrease of the calcium concentration in the basal part of the cell and an increase in the calcium concentration in the apical part of the cell. The mitochondrial calcium concentration was not significantly altered. In addition, increased sodium and decreased potassium concentrations, giving rise to a significant increase in Na/K ratio were observed in all cell compartments measured, except in the zymogen granules.
Animals↗
Nuclear zinc in human epididymal and ejaculated spermatozoa.
Sperm nuclear zinc content (expressed as zinc/phosphorus and zinc/sulphur ratios) was determined with X-ray microanalysis in individual, air-dried, epididymal spermatozoa from elderly men, and in ejaculated spermatozoa from healthy donors. Ejaculated sperm nuclei contained (also after treatment with sodium dodecyl sulphate) more zinc than epididymal spermatozoa. The results indicate that the human spermatozoon accumulates zinc from the prostatic fluid upon ejaculation.
Adult↗