On the residual six-fold astigmatism in DCOR/ASCOR.

After the introduction of the hexapole Cs-correctors for scanning transmission electron microscopes (STEM), the next big step forward was the strong reduction of the six-fold astigmatism A5 by means of an advanced hexapole design (DCOR/ASCOR). As a result all axial aberrations up to fifth order are sufficiently small to allow for large semi-aperture angles beyond 40 mrad for electron energies in the range of 30 to 300 kV without deterioration of the STEM resolution. In this paper we derive simple expressions for the optimum hexapole strength for minimum A5 and the size of the residual A5. Both quantities are intrinsic properties of the hexapoles and the transfer lens doublet in between. The optimum hexapole strength scales with the inverse of the electron wavelength, while the residual A5 does not depend on the electron energy directly, but on the spherical aberration Cs of the pole piece. With the given properties of the DCOR/ASCOR and typical values of Cs in the range of 0.5 to 2.7 mm, at all acceleration voltages A5 remains in the range from 0.03 to 0.4 mm, the latter even for a large-gap pole piece.

High-energy resolution X-ray, gamma and electron spectroscopy with cryogenic detectors.

Cryogenic detectors offer remarkably better energy resolutions than those achievable with conventional semiconductor or scintillation detectors. With the additional asset of a detection efficiency close to unity for low-energy X-ray photons and electrons, these detectors have the potential to perform X-ray, gamma and electron spectroscopy of a hitherto unknown quality, in particular at low energies. Two types of cryogenic detectors are described and the results of prototype detectors are presented.

Rat gastric hydrophobic barrier: modulation of phosphatidylcholine molecular species by dietary lipids.

Phospholipids protect the gastric mucosa by forming a proton-repellent hydrophobic layer on its luminal surface. We have recently shown that two molecular species of phosphatidylcholine (PC), PC16:0/18:1, and PC16:0/18:2, but not PC16:0/16:0, are predominantly released into gastric mucus. We investigated whether these molecular species in mucus are modified by dietary fat. Rats were fed (for three weeks) a diet supplemented with either 10% cod liver, palm, or sunflower oil, or 10% corn starch as a control. In tissue, cod liver oil decreased PC16:0/20:4 and PC18:0/20:4. Cod liver oil and palm oil increased PC16:0/18:1, whereas sunflower oil decreased PC16:0/18:1. Palm oil additionally decreased PC16:0/18:2, whereas the other diets had no effect on PC16:0/18:2. In mucus, however, PC16:0/18:1 and PC16:0/18:2 were not significantly altered by any diet. They were increased over tissue values and comprised 37.6 +/- 3.3 and 33.1 +/- 1.4 mol% in controls. PC16:0/16:0 was lower in mucus than in mucosa and even decreased by cod liver oil (1.2 +/- 0.2 vs. 2.7 +/- 0.3 mol%; P < 0.01). We conclude that PC16:0/18:1 and PC16:0/18:2 are modified by dietary fat in tissue. In gastric secretions, however, PC16:0/18:1 and PC16:0/18:2 are kept constant and together comprise 70 mol% of the released PC species, whereas PC16:0/16:0 does not play a role for the gastric hydrophobic barrier under any dietary treatment. Additionally, cod liver oil decreases the content of PC16:0/20:4 and PC18:0/20:4 in gastric mucosa, thereby possibly decreasing the formation of eicosanoids.

Composition of phospholipid classes and phosphatidylcholine molecular species of gastric mucosa and mucus.

Phospholipids have been proposed to protect the gastric mucosa by forming a proton-repellant hydrophobic layer on the gastric luminal surface, acting as a so-called gastric surfactant. The composition of this hydrophobic phospholipid layer has not previously been analysed in detail. Therefore, we measured the composition of phospholipid classes and phosphatidylcholine (PC) molecular species in gastric mucosa and mucus of rats and pigs using high resolution HPLC techniques. The predominant phospholipids of both mucosa and mucus were PC and phosphatidylethanolamine (PE). Little phosphatidylglycerol was present. The most abundant PC species of rat mucosa were PC16:0/18:1, PC16:0/18:2, PC16:0/20:4 and PC18:0/20:4. Pig mucosa also contained PC16:0/18:1, PC16:0/18:2, and PC18:0/20:4, but was poor in PC16:0/20:4. Dipalmitoyl-PC (PC16:0/16:0), the surface-active component of pulmonary surfactant, comprised only 6.42 +/- 0.33% of total PC in rat mucosa and only 5.50 +/- 1.46% of total PC in pig mucosa. Gastric mucus, isolated from both rat and pig, contained largely PC16:0/18:1 and PC16:0/18:2. The content of PC16:0/16:0 was even lower in mucus than in mucosal PC (rat 2.86 +/- 0.40%, P < 0.01; pig 1.92 +/- 0.55%, P < 0.05). We conclude that, in contrast to pulmonary surfactant, any surfactant function of the hydrophobic barrier of the stomach is unlikely to be mediated by PC16:0/16:0.

High-performance liquid chromatographic analysis of phospholipids from different sources with combined fluorescence and ultraviolet detection.

An isocratic high-performance liquid chromatographic (HPLC) system was developed for the separation of major phospholipid classes, i.e., phosphatidylcholine, sphingomyelin, lysophosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. Phospholipids were detected with ultraviolet absorption at 205 nm and subsequent fluorescence detection. Fluorescence of the phospholipids (excitation, 340 nm; emission, 460 nm) was achieved by postcolumn formation of mixed micelles with 1,6-diphenyl-1,3,5-hexatriene. For ultraviolet absorption there were great differences depending on the saturation of phospholipid fatty acids but for fluorescence the sensitivity was almost identical for all phospholipids except phosphatidylinositol and lysophosphatidylcholine. Dipalmitoylphosphatidylcholine showed nearly no ultraviolet but good fluorescence response. Ultraviolet to fluorescence ratio was characteristic for different phospholipids and for identical phospholipids from different sources. Quantification of phosphatidylcholine and phosphatidylethanolamine with HPLC using N-monomethylphosphatidylethanolamine (dioleoyl) as an internal standard gave the same results as phospholipid phosphorus quantification after thin-layer chromatography.

Phospholipid synthesis in isolated porcine gastric mucous cells.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the major phospholipids of the gastric mucosal surface barrier and chiefly originate from mucous cells. Among these phospholipids PC with palmitic acid as its hydrophobic moieties is believed to protect the gastric mucosa by its hydrophobic properties. We investigated the phospholipid synthesis of isolated porcine gastric mucous cells in vitro and incubated them in the presence of radiolabelled precursors. Incorporation of 3H-choline into PC and of 14C-ethanolamine into PE was linear at 1, 10, and 100 mumol/l substrate concentration for at least 6 h. Half-maximal rate of precursor incorporation was achieved at 21 and 15 mumol/l of choline and ethanolamine, respectively. Ethanolamine inhibited PC synthesis and choline inhibited PE synthesis. A small amount of radioactivity originating from 14C-ethanolamine and from the methyl groups of 3H-methionine were incorporated into PC. Palmitic acid was incorporated into PC more than PE. Indomethacin did not influence the de novo synthesis of PC and PE via the Kennedy pathway, but inhibited the incorporation of 3H-methionine into PC. These results indicate that in gastric mucous cells PC and PE synthesis de novo depends on the concentrations of choline and ethanolamine. The palmitic acid content of PC depends on the availability of palmitic acid as a substrate: indomethacin-induced mucosal damage is not explained by modulation of phospholipid synthesis de novo.

Isolation, identification and quantitative evaluation of specific cell types from the mammalian gastric mucosa.

Functional in vitro studies with isolated gastric mucosal cells require cytological identification of different cell types in suspension or primary culture. Since suitable techniques have not been well established, different staining methods for the discrimination of dispersed pig and guinea pig gastric cells have been developed on the basis of modified previous protocols for enzymatic cell dispersion. Chief and parietal cells were visualized by combined periodic acid-Schiff stains. Surface mucous and mucous neck cells were identified by affinity-labelling, using lectins with selective staining properties in situ. Two of the lectins were found to be specific markers for gastric polymorphonuclear cells. The following vital tests were found to be useful: succinic dehydrogenase for parietal cells, Nile blue/brilliant cresyl blue stains for chief cells, and different phagocytosis assays for endothelial cells and gastric phagocytes. Endocrine cells were characterized by immunocytochemistry using specific antibodies against gastrin, somatostatin, histamine and serotonin. The same technique using a vimentin antibody was performed for the identification of fibroblasts. Proliferation of mucosal cells in primary culture was monitored by the incorporation of bromo-deoxyuridine, which was subsequently detected by a monoclonal antibody.