W.C. de Bruijn

Ultrastructural polysaccharide localization in calcifying and naked cells of the coccolithophoridEmiliania huxleyi

SummaryEmiliania huxleyi is a marine coccolithophorid which produces coccoliths,i.e., particles consisting of calcite and macromolecular organic material. The coccoliths are formed intracellulary in specialized organelles which comprise a coccolith vesicle (CV) and a reticular body (RB), together forming the CV/RB system or calcifying system. After termination of calcification, the coccolith is extruded and incorporated into the coccosphere,i.e., one or several layers of extracellular coccoliths surrounding the cell. Apart from the coccolith-producing cells (C cells) ofE. huxleyi, there are naked cells (N cells) which seem to have lost the capacity to produce coccoliths but are very similar to the C cells in other morphological respects. Biochemical studies have revealed that polysaccharides may play a regulatory role in calcification. The aim of the present study was to determine the localization of polysaccharides in both C and N cells electron microscopically. For this purpose, a cytochemical staining technique according toThiéry (1967) was applied. The CV/RB system of C cells was conspicuously stained. Due to the excellent stainability of this system, a putative succession of morphological stages during coccolithogenesis could be described. The staining pattern of the N cells closely resembled that of the C cells. It was found, however, that the “calcifying” system of N and C cells differed in both morphology and position. It is suggested that the divergent morphology of the “calcifying” system of N cells accounts for its failure to produce coccoliths.

X-ray microanalysis of aldehyde-fixed glycogen contrast-stained by OsVI . FeII and OsVI . RuIV complexes.

: The composition of the contrast-donating complex of rat liver glycogen, nucleoplasm, erythrocytes, and mitochondria was established by X-ray microanalysis. In these compartments the presence of osmium and iron was shown qualitatively in tissue after glutaraldehyde fixation, treated with OsVIIIO4 plus K4FeII(CN)6 and in similar tissue treated with a combination of K2OsVIO4 plus K4FeII(CN)6. Osmium and ruthenium were detected in these compartments, in aldehyde-fixed tissue treated with mixtures containing K2RuIVL(CN)6 rather than K4FeII(CN)6. The iron detected in the glycogen, nucleoplasm, erythrocytes, and mitochondria of tissue treated with K2RuIV(CN)6 mixtures proved to derive from sources inside the electron microscope, and had to be considered an artifact. Quantitatively, the mean atomic ratios of osmium-to-iron and osmium-to-ruthenium were determined from spectra obtained by point analyses of the same compartments (glycogen, nucleoplasm, mitochondria, lipid droplets, and erythrocytes). After correction of the spectra for the instrumental iron contribution, the osmium-to-iron and osmium-to-ruthenium ratios in the glycogen were about 1:3 for tissue treated with those combinations including K2OsVIO4. In the other compartments, the osmium-to-iron and osmium-to-ruthenium ratios were virtually 1:0. For Os-VIIIO4 in combination with potassium ferrouscyanide however the osmium-to-iron ratio was 1:7 in the glycogen and 1:5 in all other compartments. OsVIIIO4 was combined with potassium ruthenium-cyanide, the osmium-to-ruthenium ratio was 1:2 in the glycogen and 2:1 in the other compartments. These results support our view that the selective glycogen contrast is obtained by complex formation.The composition of the contrast-donating complex of rat liver glycogen, nucleoplasm, erythrocytes, and mitochondria was established by X-ray microanalysis. In these compartments the presence of osmium and iron was shown qualitatively in tissue after glutaraldehyde fixation, treated with OsVIIIO4 plus K4FeII(CN)6 and in similar tissue treated with a combination of K2OsVIO4 plus K4FeII(CN)6. Osmium and ruthenium were detected in these compartments, in aldehyde-fixed tissue treated with mixtures containing K2RuIVL(CN)6 rather than K4FeII(CN)6. The iron detected in the glycogen, nucleoplasm, erythrocytes, and mitochondria of tissue treated with K2RuIV(CN)6 mixtures proved to derive from sources inside the electron microscope, and had to be considered an artifact. Quantitatively, the mean atomic ratios of osmium-to-iron and osmium-to-ruthenium were determined from spectra obtained by point analyses of the same compartments (glycogen, nucleoplasm, mitochondria, lipid droplets, and erythrocytes). After correction of the spectra for the instrumental iron contribution, the osmium-to-iron and osmium-to-ruthenium ratios in the glycogen were about 1:3 for tissue treated with those combinations including K2OsVIO4. In the other compartments, the osmium-to-iron and osmium-to-ruthenium ratios were virtually 1:0. For Os-VIIIO4 in combination with potassium ferrouscyanide however the osmium-to-iron ratio was 1:7 in the glycogen and 1:5 in all other compartments. OsVIIIO4 was combined with potassium ruthenium-cyanide, the osmium-to-ruthenium ratio was 1:2 in the glycogen and 2:1 in the other compartments. These results support our view that the selective glycogen contrast is obtained by complex formation.

X-ray microanalysis of colloidal-gold-labelled lysosomes in rat liver sinusoidal cells after incubation for acid phosphatase activity

SummaryThe lysosomal apparatus of the Kupffer and endothelial cells of the sinusoidal lining of the rat liver was found to take up colloidal-gold particles with a mean diameter of 5 nm, prepared according to a modified method. After incubation of the glutaraldehyde-perfusion-fixed tissue in a lead-containing medium for the demonstration of acid phosphatase activity, a reaction product was observed in the gold-loaded lysosomes. By X-ray microanalysis of such lysosomes, the presence of osmium, gold and lead was detected qualitatively in the unstained sections from the tissue, which after the incubation had been post-fixed with an OsO4-solution to which K4Fe(CN)6 had been added to enhance the contrast. The quantitative computer-assisted processing of the X-ray microanalytical data from such lysosomes enabled to determine the gold-to-lead ratio and the individual gold and lead peak intensities derived from both the Mα and Lα values in the spectra. On the basis of these results and those obtained similarly in control lysosomes containing either only gold or only lead phosphate precipitate, it was found that only the Lα values were reliable, whereas the Mα values from the same lysosomal spectra were unrealistic, due to deconvolution problems in the computer programs applied. Based upon the Lα values it was found that among the population of lysosomes in single Kupffer cells, studied after a 60-min interval between the injection of the gold colloid and fixation, three types of lysosomal contents could be quantitated by X-ray microanalysis, viz. one type with only gold, one with only lead, one with gold and lead, in various ratios. This quantitative approach might make it possible to detect variations in lysosomal composition associated with ageing.

Quantitative electron spectroscopic imaging in bio-medicine: Evaluation and application

Electron spectroscopic imaging (ESI) with the energy‐filtering transmission electron microscope enables the investigation of chemical elements in ultrathin biological sections. An analysis technique has been developed to calculate elemental maps and quantitative distributions from ESI sequences. Extensive experience has been obtained with a practical implementation of this technique. A procedure for more robust element detection has been investigated and optimized. With the use of Fe‐loaded Chelex beads, the measurement system has been evaluated with respect to the linearity of the element concentration scale, the reproducibility of the measurements and the visual usage of image results. In liver specimens of a patient with an iron storage disease the detectability of iron was tested and we tried to characterize iron‐containing components.  The concentration measurement scale is approximately linear up to a relative section thickness of ≈ 0.5. Monitoring of this parameter is therefore considered to be important. The reproducibility was measured in an experiment with Fe‐Chelex. The iron concentration differed by 6.4% between two serial measurements. Element distributions are in many applications interpreted visually. For this purpose the frequently used net‐intensity distributions are regarded as unsuitable. For the quantification and visual interpretation of concentration differences mass thickness correction has to be performed. By contrast, for the detection of elements the signal‐to‐noise ratio is the appropriate criterion.  Application of ESI analysis demonstrated the quantitative chemical capabilities of this technique in the investigation of iron storage diseases. Based on an assumed ferritin iron loading in vivo, different iron components can be discerned in liver parenchymal cells of an iron‐overloaded patient.

Mitochondrial calcium of intact and mechanically damaged bone and cartilage cells studied with K-pyroantimonate

SummaryCellular cation was localized with K-pyroantimonate osmium fixation in whole fetal mouse metatarsal bones and in deliberately mechanically damaged specimens. X-ray microprobe analysis of ultrathin sections showed a positive correlation between the concentration of Ca (and Sb) and the amount of electron-dense precipitate.In non-damaged osteoblasts and growth-plate chondrocytes dense precipitate had accumulated along the plasmalemma and the mitochondrial membranes, whereas damaged cells showed the precipitate on round granules in the mitochondrial matrix but not on membranes. Intermediate stages between these two patterns were also found.In a non-calcifying tissue such as liver no membrane-bound precipitate was found in intact cells. However, damaged liver cells showed precipitate-containing mitochondrial granules similar to those in damaged bone cells, but only after incubation of the damaged tissue for l h in a Ca-containing balanced salt solution.Freezing of fresh whole bones in liquid N2 before fixation in K-pyroantimonate osmium did not change the precipitate pattern in the damaged cells, but in intact cells it produced a random distribution of precipitate unrelated to membranes.The results are compared with those obtained in other studies on the subcellular localization of calcium and in biochemical studies on membrane versus matrix loading in calcium-accumulating isolated mitochondria.

Cytochemical and X-ray microanalysis studies of intracellular calcium pools in scale-bearing cells of the coccolithophoridEmiliania huxleyi

SummaryEmiliania huxleyi is a coccolithophorid with a life cycle including a stage characterized by the occurrence of a scale-bearing cell type. The scales are composed of organic material and are produced in the cisternae of the Golgi apparatus. The present report deals with the ultrastructural calcium localization in scale-bearing cells using cation-precipitating agents. Cations were precipitated either with potassium pyroantimonate alone or according to a combined procedure in which cells are treated first with potassium oxalate, or potassium carbonate, or potassium phosphate, and then with potassium pyroantimonate. The distribution of electron-opaque deposits was the same when visualized by all four techniques. The most extensive deposits occurred in the Golgi apparatus, the “peripheral space” (a cellular compartment totally encompassing the protoplast), the multivesicular bodies, and the cell vacuole. X-ray microanalysis revealed that calcium was a constituent of the electron-opaque deposits. The uptake and transport of calcium, as universal functions of the Golgi apparatus, are discussed.

Ultrastructural findings on lipoproteinsin vitro and in xanthomatous tissue

SynopsisThe application of OsO4 plus K3[Fe(CN)6] as a secondary fixative following aldehyde fixation, permitted demonstration of the presence of 30–300 nm ‘membrane-bound’ particles in xanthomatous tissue.With the same fixation method, isolated low density lipoprotein particles in a fibrin matrix could be observed in the transmission electron microscope in a way permitting comparison with similarly fixed tissue. However, isolated particles of very low density lipoproteins treated in the same way as low density particles had an irregular appearance and a diameter varying between 30 and 80 nm.

Scalable network monitors for high-speed links: a bottom-up approach

Monitoring traffic on high-speed links using commodity hardware is difficult due to relatively slow buses and memories. It is possible to alleviate the burden on these resources by pushing down packet processing to programmable NICs. Until now, however, the use of such cards for monitoring by network administrators has not been a practical solution, because programming the cards is too complex. For this purpose, we introduce NIC-FIX, a monitoring framework for network processors that scales to high link rates and is easy to use.

Descriptive Light and Electron Microscopy of Normal and Clue-Cell-Positive Discharge

In women with clue-cell-positive discharge (CCPD), light-microscopical examination of the wet mount suggests a preference of bacteria for certain vaginal epithelial cells (VECs). To investigate this further, a light- and electron-microscopical study of patients and healthy controls was performed, with special emphasis on vitality and glycogen content of VECs and bacterial-epithelial cell interaction. Our study did not reveal morphologic differences between VECs of patients and controls. There was, however, a significant decrease in the percentages of vital and glycogen-containing VECs in CCPD (p less than 0.001), probably caused by an overgrowth of (anaerobic) bacteria. In CCPD vaginal bacteria preferably colonize vital VECs. This could account for the relatively low percentage of clue cells in this condition.

Studies on ferritin in rat liver and spleen during repeated phlebotomy

1. The ferritin content of liver and spleen in normal and iron-loaded rats decreased during repeated phlebotomy. 2. During increased iron demand, ferritin is degraded in toto. 3. With the ESI and EELS technique the iron distribution was followed in different cell types and cellular compartments. 4. We have demonstrated two methods of iron mobilisation: (a) catabolism of lysosomal ferritin in toto and (b) delivery of ferritin from parenchymal cell into the bile and degradation of ferritin in toto.