Godfried M. Roomans

Electron probe X-ray microanalysis of human muscle biopsies

SummaryThe elemental composition of human muscle fibres have been determined by electron probe microanalysis. In order to distinguish between different types of fibres, two approaches were used. In one approach individual fibres were isolated, portions of them used for a typing by histochemical methods and the main part used for X-ray microanalysis. In the other approach the muscle biopsy was serial-sectioned, some sections used for a histochemical typing and the others (16 μm thick cryosections) used for X-ray microanalysis in the electron microscope.The comparison of the ratios between P, S and K in Study No. 1 and 2 indicates different concentrations of sulphur in the subsarcolemmal zone and in the interior of the fibre. Both routes give information on all elements (except the ten lightest ones) contained in the fibres or in sections of them, provided the concentration is high enough. In order to obtain quantitative data, expressed as mmol/kgdw, the spectra of the specimens were compared to those of standards of known composition and the data subjected to a so called ZAF-correction (corrections for the atomic number effect, absorption of X-rays in the specimen and secondary fluorescence). Quantitative data concerning phosphorus, sulphur, chlorine and potassium were obtained in Study No. 2. A significantly higher sulphur concentration was found in type IIA muscle fibres as compared to those of type I.

Accumulation of Aluminium by Anabaena cylindrica into Polyphosphate Granules and Cell Walls: an X-ray Energy-dispersive Microanalysis Study

Summary: X-ray microanalysis of thin cryosections of the cyanobacterium Anabaena cylindrica showed that aluminium was rapidly taken up and accumulated into polyphosphate granules. In addition, aluminium was found in the cell walls but could not be detected in the cytoplasm. The concentration of phosphorus in the medium affected the accumulation pattern; more aluminium was bound in the polyphosphate granules and in the cell walls after growth in phosphorus-rich medium. The accumulation of aluminium in these structures may function as a detoxification mechanism. Treatment with aluminium for 24 h did not cause any significant changes in the elemental composition of polyphosphate granules or cell walls.

Proton and Electron Microprobe Analysis of Human Skin

Abstract In order to demonstrate the feasibility of the proton microprobe in the analysis of dermatological material when a spatial resolution of a few micrometres is sufficient and to compare it with the electron microprobe technique, duplicate sections of human skin have been analysed with both methods. A skin sample was obtained from each of three healthy volunteers. After cryosectioning (12 μm) and freeze-drying adjacent sections of each sample were scanned by the electron microprobe and the proton microprobe, respectively.

Zinc in Sperm Chromatin and Chromatin Stability in Fertile Men and Men in Barren Unions

The stability and the content of zinc of the chromatin were studied in spermatozoa from ten men with unexplained infertility, and in spermatozoa from five fertile donors. A positive relation was found between zinc in sperm nuclei (X-ray microanalysis) and the resistance of the chromatin to decondense in sodium dodecylsulfate (SDS). The infertile men had lower degree of sperm chromatin stability and lower sperm zinc content than the fertile donors. A subgroup of the infertile men, which all had minor clinical signs of prostatic inflammatory reaction, had the lowest content of zinc in the chromatin and the lowest degree of chromatin stability. A low content of nuclear zinc would impair the structural stability of the chromatin and thereby increase the vulnerability of the male genome. This mechanism may be one explanation for the reduced fertility of the men with minor inflammation of the prostate.

Quantitative X-ray microanalysis of semi-thick cryosections

SummaryMethodological aspects of quantitative X-ray microanalysis of semi-thick cryosections (2–6 μm) of biological soft tissue were investigated. The preparation of a low background specimen holder is described. Scanning and scanning transmission images of the sections could be obtained, allowing identification and separate analysis of nuclei and cytoplasm. Parallel observations of histochemically stained adjacent sections in the light microscope allowed correlation of the microanalytical data with tissue morphology and histochemistry. Quantitative analysis could be carried out with the help of a standard: a gelatin/glycerol matrix containing mineral salts in known quantities, frozen and sectioned in the same way as the specimen. Mass loss under the electron beam was found to be comparable in specimen and standard. Comparison of various theoretical models for quantitative analysis showed that the ‘P/B-method’ (determination of the background intensity under the characteristic peak) is the most suitable for semi-thick sections. Factors determining the choice of accelerating voltage were analyzed. The usefulness of this specimen type is illustrated in some biological applications (human oral mucosa, rat salivary gland).

Localization of Absorbed Cadmium in Fucus vesiculosusL. by X-ray Microanalysis

Summary The localization of absorbed cadmium in the brown seaweed Fucus vesiculosus L. was studied by X-ray microanalysis in the electron microscope. The main part of the absorbed metal was found in physodes and cell walls in the outer cell layers. The distribution of metals in algae exposed to the metals in cultures under laboratory conditions was similar to that in algae growing at sites with high concentrations of heavy metals in the sea water.

The Reserpinized Rat in the Study of Cystic Fibrosis: X-ray Microanalysis of Submandibular Gland and Pancreas

The chronically reserpinized rat has been suggested as an animal model for cystic fibrosis. X-ray microanalysis of thick and thin cryosections was carried out to assess elemental redistribution in the submandibular glands and the pancreas of reserpinized rats at the cellular and subcellular level. In the submandibular gland of reserpinized rats, calcium and magnesium concentrations were significantly elevated. Mucus globules, secretory granules, and endoplasmic reticulum were the primary sites of the localization of excess calcium and magnesium. A significant potassium loss from the gland had occurred, particularly from the serous cells. Electron microscopy of conventionally prepared tissue showed marked swelling of the endoplasmic reticulum, especially in mucous cells. The elemental changes in the pancreatic acinar cells of reserpinized rats were reminiscent of elemental redistribution connected with cell death: increased levels of sodium, chlorine, and calcium and decreased levels of magnesium and potassium. Ultrastructural changes included swelling of the endoplasmic reticulum and obstruction of the acinar lumen. It is concluded tha elemental redistribution in chronically reserpinized rats presents interesting parallels with cystic fibrosis.

Calcium binding to the acrosomal membrane of human spermatozoa.

Summary The influence of divalent cations on the structure of the plasma membrane and the inner and outer acrosomal membranes of human ejaculated spermatozoa was investigated by electron microscopy. Calcium and strontium ions produced identical effects on the outer acrosomal membrane, namely an increase in thickness by about 20%, but did not change the thickness of the plasma membrane and the inner acrosomal membrane. Magnesium ions did not affect the thickness of any of the membranes. Averaged membrane profiles of the outer acrosomal membrane showed a marked difference between membranes fixed in the presence of calcium and the control. Electron microprobe analysis revealed an increase of the amount of calcium in the acrosomal region of the calcium-treated spermatozoa. It is concluded that the changes in membrane structure are due to the binding of calcium ions to the outer acrosomal membrane, indicating that the site of action of calcium on the acrosome reaction is located on the outer acrosomal membrane.

Use of pure carbon specimen holders for analytical electron microscopy of thin sections.

The use of a simple specimen holder, made of pure carbon, for analytical electron microscopy of sections of biological material is reported. Interference of the specimen holder with the X-ray spectrum from the specimen is minimized. The contribution of the holder to the continuum spectrum is consistent with theoretical predictions.

Elemental analysis of histochemically defined cells in the earthworm Lumbricus terrestris

SummaryA method for preparation of biological specimens for electron probe X-ray microanalysis is described, that aims at retaining the original elemental distribution within the tissue at the cellular level. The tissue is without any chemical fixation, quench-frozen, and 16-μm sections are prepared with a conventional cryomicrotome, transferred to a carbon specimen holder and freeze-dried.Adjacent serial sections, collected on glass slides and stained with various histological procedures, are used to correlate the data obtained by X-ray microanalysis with other histochemical information on the same cell or tissue.To demonstrate the possibilities of the method, sections of the earthworm Lumbricus terrestris were analyzed. In the chloragogenous cells, high concentrations of Ca, Zn and P were found. The inner and outer muscle layer show slightly different properties, both with regard to elemental composition and to myofibrillar ATPase activity.