X-ray Microanalysis Of Cryosections Research Articles

Cellular Calcium Distribution Modulates the Growth of Callus and Protoplasts of Halophyte Mangrove Plant, Avicennia Alba - an X-ray Microanalysis

Two cultured cell lines were developed from cotyledons of a halophyte mangrove, Avicennia alba.In the high-Ca callus line, which was sub-cultured in amodified amino acid medium containing 3 mM CaCl2, growth of calluses and their protoplasts were both inhibited by low concentrations of CaCl2 in the culture medium. Removal of Ca2+ from the culture medium stimulated callus growth and the calluses could be sub-cultured without CaCl2 (low-Ca callus line). The intra- (cytoplasmic matrix and vacuole) and extra- (cell wall) cellular concentrations of elements, i.e., [Ca], [K], [Cl], [Na], [Mg], [P] and [S] were investigated using quantitative X-ray microanalysis of cryosections of calluses from bothcell lines. [Ca] was high in the cytoplasmic matrix and cell wall of the high-Ca line. [Ca] was lowered in the low-Calineinall cell compartments, though still detected. Ca-containing electron-dense precipitates were accumulated in the middle lamella of cell walls in resin-embedded sections of the high-Ca line. CaCl2 in the medium stimulated protoplast growth only in the low-Caline. These results suggested that a low cellular [Ca] is needed for protoplasts growth of A. alba. The importance of cellular [Ca] for the growth of halophilic mangrove plant cells was discussed.

Cytochemical and electron probe X-ray microanalysis studies on the distribution change of intracellular calcium in columella cells of soybean roots under simulated microgravity

The columella cells of soybean roots grown under gravity and simulated microgravity induced by a clinostat were examined using potassium pyroantimonate (PA) and quantitative X-ray microanalysis of cryosections to determine the role of Ca in the regulation of the gravitropic response. Amyloplasts in the columella cells were localized exclusively at the bottom under gravity, but diffusely distributed in the cytoplasmic matrix under simulated microgravity, thus supporting the statolith theory. In the columella cells, PA precipitates containing Ca were diffusely distributed in the cytoplasmic matrix under gravity. Under simulated microgravity, however, they decreased in number and size in the cytoplasmic matrix, whereas increased only in number in the vacuole, indicating that Ca moved from the cytoplasmic matrix into the vacuole. The vacuole of columella cells contained mostly electron-dense granular structures localized along the inner surface of tonoplasts, which closely resembled the tannin vacuole reported in Mimosa pulvinar motor cells. Under simulated microgravity, their configuration changed dramatically from a granular shape to a flat plate. The quantitative X-ray microanalysis of cryosections showed that the vacuolar electron-dense structures contained a large amount of Ca. Under simulated microgravity, the concentration of Ca increased conspicuously in these vacuolar electron-dense structures, concomitantly with a marked decrease of K in the vacuoles and an increase of K in the cell walls. These results suggest that the release of Ca(2+) from, and uptake by, the vacuolar electron-dense structures is closely related to the signal transmission in the gravitropic response and that Ca movement occurs opposite to that of K.

LIMITATIONS AND PROSPECTS OF BIOLOGICAL ELECTRON PROBE X-RAY MICROANALYSIS

ABSTRACT Analytical electron microscopy is used to identify, localize and quantify chemical elements in biological samples with the aim to correlate the element distribution in cells and tissues with the particular functional state. Energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections is considered to be the most efficient technique with respect to spatial analytical resolution and detection limit. The potentialities and limitations of this technique are illustrated by results from 3 current research projects: Element compartmentation in hepatocytes, cytotoxicity of an organotin compound, and calcium movements during osmoregulation of epithelia. At present, the absolute detection limit is 170 atoms in a minimum analytical volume of 30 × 30 × 100 nm3. X-ray microanalysis of cryosections from biological samples is limited by ice crystal growth during cryofixation and by the poor X-ray collection efficiency in most electron microscopes. Therefore, the construction of an analytica...

Carbonic anhydrase supports electrolyte transport in Drosophila Malpighian tubules. Evidence by X-ray microanalysis of cryosections

Electron probe X-ray microanalytical studies on the role of carbonic anhydrase in electrolyte transport in the cells of Drosophila Malpighian tubules indicate that carbonic anhydrase delivers protons and bicarbonate ions to ion transport systems in the cell membrane. After injection and after feeding acetazolamide or hydrochlorothiazide, known inhibitors of carbonic anhydrase, the contents of potassium, magnesium and chloride in the apical cytoplasm and in the cytoplasm close to the basal plasma membrane decreased. We explain our measurements by the hypothesis of a basal Mg-H-antiport system in parallel with Cl-HCO 3-antiport, inhibitable by DIDS. Zinc is supposed to enters cells and intracellular Zn storage vacuoles by a negatively charged Zn-anion-complex in exchange for HCO 3 - ions. This antiport is inhibitable by SITS. The content of the Zn storage vacuoles is acid, as shown by red fluorescence after incubation of Malpighian tubules with acridine orange. Red fluorescence is absent after preincubation in a medium containing an inhibitor of carbonic anhydrase. Carbonic anhydrase was demonstrated cytochemically in the Golgi-ER complex, Golgi vesicles and intercellular space. We suppose that carbonic anhydrase is synthesized and stored in the Golgi-ER-complex from where it is released into the tubule lumen.

Evaluation of the pyroantimonate method for detecting intracellular calcium localization in smooth muscle fibers by the X-ray microanalysis of cryosections.

The validity of the pyroantimonate method, which has been used for detecting intracellular Ca localization and translocation in smooth muscles, was examined by making cryosections of the relaxed anterior byssal retractor muscle (ABRM) of Mytilus edulis at various stages of procedures for preparing ordinary Epon-embedded sections and determining the elemental concentration ratios of the pyroantimonate precipitate, localized along the inner surface of the plasma membrane, with an energy dispersive X-ray microanalyzer. The concentration of Ca (relative to that of Sb) in the precipitate stayed constant after the procedures of fixation, dehydration and Epon-embedding, while the concentrations of K, Mg, Na and Os showed their respective characteristic changes after the above procedures, being lower than that of Ca in the Epon-embedded sections. The presence of Ca in the precipitate was also demonstrated with an electron energy-loss spectrometer. The localization of Ca underneath the plasma membrane was also observed in the cryosections of the ABRM fibers prepared after mild fixation with acrolein vapor without using pyroantimonate. These results indicate that the pyroantimonate precipitate serves as a valid measure of intracellular Ca localization.

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 α-adrenergic, β-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 fibroblats 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.