Determination of electrolytes in small biological fluid samples using energy dispersive X-ray microanalysis

Summary A technique is reported for the application of the energy dispersive X-ray microanalysis for the simultaneous determination of electrolytes in picoliter samples of biological fluids. The preparation is characterized by the use of thin films as a specimen support. Using this arrangement, the X-rays generated in the support are kept to a minimum. At an acceleration voltage of 25 kV the specimen preparation can be regarded as thin, i.e. the intensity of an emitted characteristic radiation is almost uninfluenced by the gross elemental composition, and, therefore, is only dependent upon the content of elements. Quantification was achieved by comparing the intensities of the characteristic radiations with that of a standard. Electrolyte concentrations of 1 mM can be detected with an accuracy of 0.1 mM SD.Supported by the Deutsche Forschungsgemeinschaft     

Quantitative roentgen microanalysis of biological ultrathin sections with aluminum-carbon vapor films as standards

A technique is given to separate extraneous and sample background in x-ray microanalytical spectra. It is shown, that evaporated aluminium-carbon films with aluminium concentrations ranging between 1 and 10% are well suited as standards for biological x-ray microanalysis.     

Topochemistry of trace metals in nasal mucosa. Potentialities of some histochemical methods and energy dispersive X-ray microanalysis.

Histochemical methods and energy dispersive X-ray micro-analysis (EDX-analysis) were evaluated in model experiments and on tissue sections for their usefulness in detecting traces of metals in biological tissue. The goal for this study was to establish a method for localization of nickel deposits in the nasal mucosa, where it has been found in concentrations between 1 and 40 microgram/g in nickel exposed individuals. The histochemical methods tested were staining with dimethylglyoxime, rubeanic acid and dithizone, the Turnbull and Prussian blue methods and TIMM'S sulphide silver procedure. In model experiments nickel-, cobalt-, copper-, zinc- and ironsalts were applied to thin-layer chromatography sheets (TLC-sheets) and stained by the histochemical methods. Spots containing 500 and 50 ng of these metals represented the smallest amounts that could consistently be detected in these experiments, except for the sulphide silver method which seemed a little more sensitive. With the latter method, moreover, zinc was detected in 40 micrometer thick cryostat sections of gelatine made up with 1 microgram/g of the metal. For nickel the corresponding figure was 10 to 50 microgram/g. On specimens of nasal mucosa from nickel-exposed workers, a faint colour was obtained in 40 micron thick cryostat sections from specimens that had been immersed in dithizone, but the colour was too weak for histological analysis. None of the other coloured chelating agents caused noticeable staining when applied to blocks or to cryostat sections. TIMM'S sulphide silver method caused strong staining of the basal layers of the surface epithelium and of fibroblast-like cells in the underlying connective tissue. This staining pattern is described in more detail in a separate report. Rat liver tissue was analyzed by atomic absorption before and after araldite embedding. Blocks of gelatine made up with nickel, copper, zinc and iron were embedded in epoxy resin and analyzed by atomic absorption. Large changes in the metal concentrations, usually an increase, were found after embedding. Ultrathin sections from this material were used to test the sensitivity of the EDX-equipment. Referring to the concentrations determined by atomic absorption in the embedded material, iron was detected at 1215 microgram/g and 362 microgram/g (gelatine standards) but not at 167 microgram/g (rat liver). Similar values could not be determined for nickel, copper or zinc, because of background radiation resulting from the presence of these metals in the instrument. We did not succeed in establishing a procedure for detecting nickel deposits in nasal mucosa with any of the methods which were tested. The most sensitive but least specific of the tested methods for visualizing heavy metals in the nasal mucosa, was TIMM'S sulphide silver procedure. The preparation of tissue for this method is discussed.     

Tissue reactions to the separate implantation of individual constituent phases of dental amalgam,including assessment by energy dispersive X-ray microanalysis

Soft tissue degradation of the 3 principal amalgam phases have been investigated in relation to their role in the formation of the amalgam tattoo. Each phase, finely powdered, was implanted subcutaneously into the submandibular region of guinea-pigs for periods ranging from 1 week to 1 year. The rates of breakdown were assessed radiographically and the final lesions were examined by light and electron microscopy and X-ray microanalysis. γ₂ (Sn₇Hg) phase degraded rapidly, mainly extracellularly, and did not produce a tattoo. Both mercury and tin disappeared from the lesion, γ₁ (Ag₂Hg₃) phase degraded less rapidly, both extra and intracellularly, and produced a small tattoo. Mercury was lost from the lesion. γ (Ag₃Sn) phase degraded slowly, intracellularly, and produced a large tattoo. Tattoos always resulted from persistence of minute particles of silver and sulphur associated with basal lamina and connective tissue.     

Matrix crystals in cytologic urine specimens: Observations on their mineral composition by energy dispersive X-ray microanalysis and morphologic scanning electron microscopy

Crystals consisting by light microscopy of organic matrix (matrix crystals) encountered in cytologic urine specimens of 8 patients were examined for mineral phase components by scanning electron microscopy with energy dispersive X-ray microanalysis (SEM-EDX) and by morphologic scanning electron microscopy (SEM) performed separately in four of the eight cases. Whenever possible (three cases) mineralized crystals present in these specimens were examined separately by SEM-EDX for comparison of mineral phase composition with that of the corresponding matrix forms. Although by SEM-EDX components of matrix, glass and slide preparation media interfere with the precise estimation of the mineral phase components, the results of this method supported by the SEM morphology suggest that crystals consisting of organic matrix include a mineral phase, the lattice structure of which provides them from the early stages of formation with the characteristic morphology of the fully mineralized forms. This also suggests that organic matrix plays a role in the nucleation of minerals during the formation of certain urinary crystals. Diagn Cytopathol 1994; 11:38–46. © 1994 Wiley-Liss, Inc.     

A new technique to prevent rehydration of freeze-dried sections for X-ray microanalysis: Papillary muscles and myocyte specimens

There is considerable interest of the changes in intracellular electrolytes in various pathophysiological states in cardiology. X-ray microanalysis is a useful method to measure the concentration of intracellular elements, and freeze-drying is a required technique for X-ray microanalysis to preserve elements. In this study, we utilized a new technique by which ultrathin sections were evaporated by carbon in an external freeze-dryer after freeze-drying and we were able to obtain clear electron micrographs of papillary muscles and myocytes. Calcium concentrations of mitochondria and cytosol were measured during 120 min ischemia. We showed that mitochondria played a role in intracellular calcium regulation during 30 min ischemia.     

X-ray fluorescence and energy dispersive x-ray diffraction for the quantification of elemental concentrations in breast tissue

This paper presents improvements on a previously reported method for the measurement of elements in breast tissue specimens (Geraki et al 2002 Phys. Med. Biol. 47 2327-39). A synchrotron-based system was used for the detection of the x-ray fluorescence (XRF) emitted from iron, copper, zinc and potassium in breast tissue specimens, healthy and cancerous. Calibration models resulting from the irradiation of standard aqueous solutions were used for the quantification of the elements. The present developments concentrate on increasing the convergence between the tissue samples and the calibration models, therefore improving accuracy. For this purpose the composition of the samples in terms of adipose and fibrous tissue was evaluated, using an energy dispersive x-ray diffraction (EDXRD) system. The relationships between the attenuation and scatter properties of the two tissue components and water were determined through Monte Carlo simulations. The results from the simulations and the EDXRD measurements allowed the XRF data from each specimen to be corrected according to its composition. The statistical analysis of the elemental concentrations of the different groups of specimens reveals that all four elements are found in elevated levels in the tumour specimens. The increase is less pronounced for iron and copper and most for potassium and zinc. Other observed features include the substantial degree of inhomogeneity of elemental distributions within the volume of the specimens, varying between 4% and 36% of the mean, depending on the element and the type of the sample. The accuracy of the technique, based on the measurement of a standard reference material, proved to be between 3% and 22% depending on the element, which presents only a marginal improvement (1%-3%) compared to the accuracy of the previously reported results. The measurement precision was between 1% and 9% while the calculated uncertainties on the final elemental concentrations ranged between 10% and 16%.     

Electron microscopy of virus particles in ultrathin frozen sections.

A procedure with the use of polyethylene glycol (PEG) 600 to support biological materials for ultracryotomy was developed. Developing and mature virions were demonstrated by electron microscopy in negatively stained frozen sections of vaccinia virus-infected human diploid cells and of herpes simplex virus (HSV) type 1 infected primary rabbit kidney cells. Approximate localisation of the virus particles in the cells was possible.     

Pseudofungi in a lymph node. A case report with energy dispersive X-ray elemental analysis.

A case of pseudofungi in axillary lymph nodes is presented. They resembled septate hyphae and were stained with a periodic acid-Schiff stain. Gomori methenamine silver stain failed to stain them. Because of morphologic similarity to true septate fungi and positive staining with periodic acid-Schiff stain, these structures can be misinterpreted as septate hyphae forming true fungal organisms. Further studies, some of which included energy-dispersive x-ray elemental analysis and special stains, revealed that they were composed of iron, phosphorus, and calcium. The relationship of these pseudofungi to previously published cases and clues that enable one to make a correct diagnosis are described.     

X-ray microanalysis of the Michaelis-Gutmann bodies of malakoplakia

The Michaelis-Gutmann (MG) inclusion bodies of three cases of malakoplakia (prostate, testis, colon) were studied by X-ray microanalysis to determine their elemental composition. Calcium and phosphorus were consistently found. Iron was detected in a few bodies. No other elements were detected. The electrondense laminations were of similar composition to the core material. Small aggregates of electron-dense material containing calcium and phosphorus were also occasionally seen in phagolysosomes. These observations are consistent with view that MG bodies arise by a process of phagolysosomal coalescence and mineralisation.     

Immunohistochemical identification of immunoglobulin A on ultrathin tissue sections

Antigenic determinants of the heavy chain of immunoglobulin A (IgA) were identified within immunocytes by immunohistochemical techniques applied to ultrathin sections of rat ileal mucosa embedded in epoxy resin. Successful localization of IgA depended upon the utilization of chemical fixatives containing paraformaldehyde or physical fixation employing a freeze-drying technique. Partial removal of the epoxy resin from the ultrathin tissue section with metallic alkoxide solutions or pretreatment with H₂O₂ was essential prior to the sequential application of the immunological reagents. IgA was associated with the rough endoplasmic reticulum (RER) and nuclear regions of immunocytes. These results are similar to those of other investigators employing immunochemical localization before embedment.     

Characterization of acrylic polyamide plastic embolization particles in vitro and in human tissue sections by light microscopy, infrared microspectroscopy and scanning electron microscopy with energy dispersive X-ray analysis.

Vascular embolization is a well-established practice for the treatment of tumors and vascular lesions. Rounded beads (microspheres) of various materials (collagen, dextran and trisacryl-polymer-gelatin) were developed to solve problems encountered with earlier versions of embolic material. We performed histochemistry, Fourier transform infrared microspectroscopy and scanning electron microscopy with energy dispersive X-ray analysis on two uterine and one hepatic specimen with unidentified intravascular foreign material, and examined a reference embolization product for comparison. The hematoxylin and eosin stained tissue sections showed multiple foci with unidentified intravascular foreign material and fibrous obliteration of vessel lumens. Only one case had a clinical history of previous embolization but without specifying the material used. One case was submitted for identification of a 'parasite'. The material stained positively with Sirius red and mucicarmine, variably with Masson's trichrome stain and Movat pentachrome, and did not stain centrally with periodic acid Schiff with diastase. Infrared spectrophotometric analysis of the material from all three cases demonstrated the spectrum of acrylic polyamide plastic. A control sample of EmboGold exhibited infrared microspectroscopic spectra similar to the three tissue specimens. Analysis by scanning electron microscopy with energy dispersive X-ray analysis demonstrated some differences in elemental composition between the tissue sections and the selected reference material. To our knowledge, this is the first report of infrared spectrophotometric analysis with scanning electron microscopy with energy dispersive X-ray analysis of an acrylic polyamide plastic embolization product both in vitro and in human histologic tissue sections. In cases lacking appropriate clinical information, identification by these methods and/or a panel of special stains may assist pathologists unfamiliar with this material's light microscopic appearance.     

X-ray microanalysis of resting and stimulated rat pancreas

The elemental distribution in acinar cells of rat pancreas was investigated by X-ray microanalysis of thin, freeze-dried cryosections. In the resting cell, the highest calcium concentrations were found in the basal part of the cell (including the endoplasmic reticulum) and in the zymogen granules. Mitochondrial calcium concentrations were low. Zymogen granules were rich in sulphur, but low in phosphorus, sodium and potassium. Stimulation of the pancreas by perfusion in vivo with the cholinergic agonist carbachol caused a significant decrease of the calcium concentration in the basal part of the cell and an increase in the calcium concentration in the apical part of the cell. The mitochondrial calcium concentration was not significantly altered. In addition, increased sodium and decreased potassium concentrations, giving rise to a significant increase in Na/K ratio were observed in all cell compartments measured, except in the zymogen granules.     

Ultrastructural analysis of salivary calculus in combination with X-ray microanalysis

Ultrastructural studies of salivary calculi were performed. Scanning electron microscopic examination of the calculi revealed lamellar and concentric structures. Granular or globular structures and pyramid structures were found on the surface of the calculi, and in some cases a scaly structure corresponding to fiber and bacteria was recognized. X-ray microanalysis showed the main constitutes of the calculi to be Ca and P. Transmission electron microscopic examination revealed a fine fibrous structure near the degenerated organelles, and analyses of the structure by electron diffraction revealed hydroxyapatite. Calcification was found around the degenerative organelles in the form of lipid-like structures, mitochondria, lysosomes, and microbial structures. Judging from our results, as one of the processes leading to calculi formation, it is speculated that degenerative substances are emitted by saliva, by some phenomenon, and calcification around these substances then occurs, contributing to the formation of calculi.