Morphological findings and energy dispersive X-ray microanalysis of oral amalgam tattoos.

Oral amalgam tattoos (AT) are distinct pigmentations of the oral mucosa resulting from accidental incorporation of dental amalgam in the oral soft tissues. Dental amalgams and in particular mercury, one of the constituents of dental amalgams, have for long been considered toxic. Oral ATs are easily accessible to study soft tissue reaction to amalgam and its degradation products. In this study, 17 oral ATs were examined by transmission electron microscopy and energy dispersive X-ray microanalysis. Ultrastructurally, in the ATs, three kinds of electron-dense particles were observed. The largest particles ranged in size from 0.5 up to several 100 microm. Smaller electron-dense inclusions (0.5-0.1 microm) were seen extracellularly associated with meshworks of elastic fibers and collagen bundles. The third and smallest type of particles (5-30 nm in diameter) was found with basement membranes of small vessels and pericytes and particularly decorating collagen bundles. Element analysis regularly revealed the presence of silver, sulphur, copper and lead in the AT decay products. Mercury was found in only one instance. Tissue reactions due to ATs seem to be minimal. No acute inflammatory changes were seen. Larger inclusions occasionally were surrounded by macrophages and multinucleated cells. TEM and element analysis may in specific cases be helpful in the differential diagnosis of pigmented lesions of the oral mucosa.

Preparation of cells cultured on silicon wafers for mass spectrometry analysis.

The distribution of specific atoms and molecules within living cells is of high interest in bio-medical research. Laser secondary neutral mass spectrometry (laser-SNMS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) detect atoms with high sensitivity and spatial resolution. The application of these methods to cultured cells requires special preparation techniques preserving morphological and chemical integrity of the living cells. The cells should, therefore, be grown on a conducting material preventing charging of the sample during ion bombardment. Silicon is currently used as the preferred support material for non-biological samples in mass spectrometry. This study investigates (1) the influence of silicon surfaces on cell growth and (2) the suitability of a sandwiched, rapid freezing method to analyse transmembrane ion gradients. Human melanoma cells were grown on silicon with polished or etched surfaces. Growth kinetics were studied using the Sulforhodamine-B assay. Number, shape, and morphology of the cells were assessed by epifluorescence microscopy of calcein AM- and DAPI-stained cells. Cells were subjected to rapid freezing, freeze-fracturing, and freeze-drying prior to analysis by TOF-SIMS and laser-SNMS. While cell numbers and morphology on the rough silicon wafers were impaired, morphology and growth kinetics of cells on polished silicon were identical to control cells on cell culture tested polystyrene. TOF-SIMS and laser-SNMS resulted in high-resolution elemental images and mass spectra. Measurement of the intracellular Na+ and K+ concentrations revealed a ratio as observed in living cells. In conclusion, culturing cells on polished silicon wafers followed by sandwiched, rapid freezing is an adequate preparation method to study intracellular ion distribution with mass spectrometry.

Magnesium transport through the basal plasma membrane of larval malpighian tubules of Drosophila hydei studied by electron probe X-ray microanalysis.

Magnesium besides calcium is the most important excretion product. In the anterior Malpighian tubules of Drosophila, excretion of magnesium takes place via the hindgut by proteoglycan containing concretions. This study reports on magnesium transport through the basal plasma membrane of the principal cells of the proximal segment of the anterior Malpighian tubules. Measurements by electron probe X-ray microanalysis indicate the existence of two antiporters which transfer magnesium in still unknown stoichiometry from the hemolymph space into the cell: Mg/H and Mg/Na.

The distribution of light elements in biological cells measured by electron probe X-ray microanalysis of cryosections.

The intracellular distribution of the elements carbon, nitrogen, and oxygen was measured in cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of 100-nm-thick freeze-dried cryosections. Electron irradiation with a dose up to 106 e/nm2 caused no or merely negligible mass loss in mitochondria and in cytoplasm. Cell nuclei lost carbon, nitrogen, and-to a clearly higher extent-oxygen with increasing electron irradiation. Therefore, electron doses less than 3 x 105 e/nm2 were used to measure the subcellular compartmentation of carbon, nitrogen, and oxygen in cytoplasm, mitochondria, and nuclei of the cells. The subcellular distribution of carbon, nitrogen, and oxygen reflects the intracellular compartmentation of various biomolecules. Cells exposed to inorganic mercury before cryofixation showed an increase of oxygen in nuclei and cytoplasm. Concomitantly the phosphorus/nitrogen ratio decreased in mitochondria. The data suggest mercury-induced production of ribonucleic acid (RNA) and decrease of adenosine triphosphate (ATP). Although biomolecules cannot be identified by X-ray microanalysis, measurements of the whole element spectrum including the light elements carbon, nitrogen, and oxygen can be useful to study specific biomolecular activity in cellular compartments depending on the functional state of the cell.