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Electron probe X-ray microanalysis of epithelial cells: aspects of cryofixation.

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, the K/Na-ratio increased to 24. Effects of extracellular media of different composition on the intracellular element content were studied. Malpighian tubules of Drosophila larvae were cryofixed by rapid immersion into liquid propane, and the distribution of K across the cells forming the tubules from the basal to the apical cell membrane was measured. An increasing K gradient was found from the intermediate to the apical cytoplasm. The intracellular K distribution was dependent on ions and transport inhibitors present in the fluid surrounding the Malpighian tubules within the larvae. Content and distribution of ions in epithelial cells sensitively depend on the physiological state immediately before cryofixation. Thus, electron probe X-ray microanalysis of cells and cell functions requires careful selection and control of the cell system to be studied.

Animals↗

Specimen damage considerations in biological microprobe analysis.

In many biological materials radiation damage limits the resolution of the microanalytical measurement. To provide some perspective regarding the relative merits of different experimental arrangements, the dose to the specimen may be calculated using a simple model. While electron and proton probe X-ray microanalysis are found to involve heavy doses to the specimen, X-ray fluorescence, performed with a polarized, monochromatic X-ray probe, is the least destructive for the analysis of medium to heavy elements. For light elements (Z less than or equal to 20), electron energy loss spectroscopy or X-ray absorption microanalysis involve the lowest dose in most applications. Other advantages and limitations of the various techniques are also summarized.

Electron Probe Microanalysis↗

Adrenal potassium and sodium in experimental hyper- and hypoaldosteronism in the rat. Determination by electron probe x-ray microanalysis.

Potassium and sodium contents in the various adrenal zones were determined in experimental hyper- and hypoaldosteronism in the rat by electron probe X-ray microanalysis. The analysis aimed at revealing intracellular values. There was no change in the potassium content of the zona glomerulosa, zona fasciculata and medulla neither in hyperaldosteronism induced by Na-deficiency, nor in hypoaldosteronism, elicited by Na-rich diet. The sodium content in the zona glomerulosa and zona fascicularis was increased in the Na-loaded rats, while that of the medulla was not changed. Our data are not consistent with the hypothesis that a change of adrenal intracellular potassium would act as a final stimulus in the regulation of aldosterone secretion.

Adrenal Glands↗

Gentamicin ototoxicity in otoconia: quantitative electron probe X-ray microanalysis.

Chronic gentamicin ototoxicity was evaluated in the otolithic membrane of adult OF1 mice at the otoconial layer of the saccule and utricle by quantitative electron probe X-ray microanalysis of Ca and K. The otolithic membranes were plunge-frozen and freeze-dried. The analysis was carried out with an energy dispersive detector using the peak-to-back-ground ratio method and different inorganic salts of Ca and K as standards to calibrate the microprobe. Ca and K in the otoconia are related via a linear function in both the saccule and the utricle. This association is not maintained after exposure to gentamicin, which suggests that this aminoglycoside antibiotic interferes with the Ca-K equilibrium in the otoconia. A dose of 200 mg/kg gentamicin twice a day for 5 days did not affect Ca in the mineral phase of the otoconia, but did increase K in both saccular (p < 0.05) and utricular (p < 0.01) otoconia. These increases in K may reflect a modification in the composition of the endolymph, resulting from cellular damage at the plasma membrane.

Animals↗

Directed metal determination in pre- and post-natal stages of development of the islets of Langerhans by combined histochemical metal demonstration with electron probe X-ray microanalysis.

On the examples shown above it was possible to demonstrate that electron-X-ray microanalysis does not only present an important additional method for studying histochemical heavy metal reactions but can also be expected to provide, in combination with these, essential findings for the illustration of basic biological processes.

Animals↗

Electron probe X-ray microanalysis of K, Rb, Cs, and T1 in cryosections of striated muscle.

Muscles containing the normal amount of K+ or loaded with Rb+, Cs+, or T1+ were cryofixed, cryosectioned and analysed by electron probe X-ray microanalysis. Previously reported results obtained with independent methods were confirmed: The alkali-metals and T1 are mainly localized in the A bands and at Z lines of the striated muscle. The results are in accordance with the association-induction hypothesis and the concept that K+ is physically adsorbed onto beta- and gamma-carboxyl side chains of myosin and other proteins.

Animals↗

Electron probe x-ray microanalysis of sarcolemma and junctional sarcoplasmic reticulum in rabbit papillary muscles: low sodium-induced calcium alterations.

This project was undertaken to determine whether electron probe x-ray microanalysis (microprobe analysis) could be utilized to determine the subcellular sites responsible for low sodium-induced calcium accumulation in myocardium. Ultrathin cryosections of rabbit papillary muscles were analyzed using microprobe analysis, and the concentrations (mmol/kg dry wt) of Na, Mg, I, S, Cl, K, and Ca were compared against low sodium (36 mM) and control (139 mM NaCl) muscle groups. Visual resolution of junctional sarcoplasmic reticulum in freeze-dried myocardial sections was achieved, and systematic analysis of junctional sarcoplasmic reticulum and sarcolemma was performed. Myofibrils and mitochondria were also analyzed. Reductions in Na and Cl concentration were measured in virtually all compartments of muscles bathed in low sodium. In addition, low sodium produced a doubling of junctional sarcoplasmic reticulum and sarcolemmal calcium concentrations (p less than 0.01). No significant changes in calcium were observed at other analyzed sites. The increased calcium at the junctional sarcoplasmic reticulum and sarcolemma correlates with (but may not completely account for) the threefold increase in contractility measured after 40 minutes in low sodium concentrations. This work demonstrates that elemental changes associated with the myocardial junctional sarcoplasmic reticulum and sarcolemma are amenable to direct, in situ microprobe analysis and further defines these structures as primary sites of calcium accumulation in low sodium concentrations.

Animals↗

Electron probe X-ray microanalysis of rabbit ciliary epithelium.

Rabbit iris-ciliary bodies were preincubated in control and experimental Ringer's solutions before quick freezing, cryosectioning, dehydration and electron probe X-ray microanalysis. After preincubation in a baseline bicarbonate-free Cl- Ringer's solution, the ciliary epithelial intracellular Na+, K+ and Cl- concentrations were estimated to be 15 +/- 3, 162 +/- 14 and 46 +/- 5 mmol kg-1 intracellular water, respectively. The water and elemental Na, K, Cl and P contents were similar in the non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells. As expected, inhibition of the Na,K-exchange pump by preincubation with ouabain markedly increased the intracellular Na content, and markedly reduced the intracellular K content, verifying the validity of the experimental analysis. The Cl- channels of the NPE cells likely play a critical role in determining the rate of aqueous humor formation. Therefore, we have examined the effects of altering Cl- transport on the intracellular composition in this initial microprobe study of the ciliary epithelium. As expected, exposure to bicarbonate increased the intracellular Cl and water contents. Replacement of external Cl- by NO3- was twice as effective as replacement by gluconate in leaching Cl- out of the intracellular compartment. An unexpected finding was that NO3- replacement of internal Cl- substantially increased the intracellular Na and decreased the intracellular K content, possibly by stabilizing the Na,K-pump in the E1P form and inhibiting enzyme activity.

Animals↗

Quantitative Estimates of Phosphorus Concentrations within Lupinus luteus Leaflets by Means of Electron Probe X-ray Microanalysis.

Phosphorus contents of epidermal vacuoles and mesophyll cells of Lupinus lutens leaflets were measured by electron probe x-ray microanalysis of fully hydrated, bulk frozen samples. Quantitation was achieved using standard solutions containing colloidal graphite to simulate cell contents and the derived nonlinear relationship between peak over background ratio and concentration improved the accuracy of the analytical procedure. Inorganic phosphate contents of mesophyll cells were shown to be highly dependent on phosphate nutrition. Comparison with data obtained from conventional analysis leads to the suggestion that in heterogeneous tissues the inorganic phosphate concentrations of the cytoplasm may show greater variation than observed in the cytoplasm of simple plant systems such as cell suspension and root tips.

Journal Article↗