Romuald Wroblewski

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.

Elemental Composition of the Developing Inner Ear

The elemental composition of the inner ear fluid-filled compartments has been analyzed using the x-ray energy dispersive technique (CBA mouse). Special attention has been focused on the maturation of endolymph. A few days before and after birth the relative peak intensity of potassium (R k ) in the vestibular endolymphatic compartment was slightly surpassing that in the cochlear part of the labyrinth. From the fourth day after birth (DAB) a rapid increase occurred concerning the R k . The highest R k was found in the endolymphatic space in the basal part of the cochlea followed by that in the vestibular endolymph. The lowest R k was measured in endolymph of the apical part of the cochlea. These obvious differences were abandoned already on the sixth DAB. A mature composition of endolymph was reached on the eighth DAB. The present technique does not allow analyses of differences between cochlear and vestibular endolymph with regard to minimal shifts.

Elemental Composition of Individual Cells and Tissues in the Cochlea

Localization of elements at the cellular and sub-cellular levels was performed with the energy dispersive X-ray microanalysis technique, using shock-frozen, freeze-dried and araldite-embedded mouse (CBA/CBA) cochleae sectioned dry. Anatomical identification occurred in the STEM (scanning transmission electron microscopy) mode. In inner hair cell stereocilia the K/Na ratio was 70:1 but only 20:1 in the cytoplasm. In outer hair cell cytoplasm the K/Na ratio was 11:1 while the ratio in stereocilia was similar to that in inner hair cells. Ca was identified in stereocilia and the upper part of the cytoplasm of both outer and inner hair cells. The elemental composition in the subtectorial space is endolymph-like and that in the inner tunnel of the organ of Corti is similar to extracellular fluid. Considerable regional differences in elemental composition occur in the tectorial membrane with regard to P, K and Ca. The highest concentration of Ca occurs in the basal part of the tectorial membrane towards the sensory hairs. The highest concentration of K occurs in the basal and outer parts whereas the middle part of the tectorial membrane contains low levels of both K and Ca. The elemental composition changes in two main directions: 1) from the limbal (growing) region to the tip of the tectorial membrane, and, 2) from upper to lower surfaces. The three cell types of the stria vascularis differ considerably in elemental composition. The highest concentration of K occurs in marginal cells. The basal cells contain more K than do the intermediate cells. A significantly higher concentration of Ca, Cl and Na occurs in marginal cell cytoplasm than in any other cell type in the stria vascularis.

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).

Elemental Composition of the Mature Inner Ear

An X-ray energy dispersive analysis was performed on the mature serial sectioned freeze-dried mammalian labyrinth (CBA/CBA mouse). The elemental distribution in the endolymphatic and perilymphatic spaces revealed similar results as have been reported in the literature when withdrawing fluid for microanalysis. The cupulae of the vestibular organs showed only minimal relative peak intensities of chlorine and potassium in contrast to the tectorial membrane in the cochlea with elemental identification also of sodium, sulphur and calcium. A difference in structural composition is therefore likely between these two tissues.

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.

X-Ray microanalytic studies on developing otoconia

The onset of otoconial development in the macula utriculi in CBA/CBA mice occurs on the fifteenth and sixteenth gestational days. Our study was concentrated on the early secretion of calcium in the areas of otoconial formation. The epithelial origin of otoconia is documented. Protrusions from supporting cells in the utricular and saccular epithelia contain very large quantities of calcium in addition to the normal cytoplasmic content of elements. The cell protrusions have an elemental composition clearly differing from that of otoconia as well as from neighbouring utricular cells with protrusions. A directed flow of calcium to developing otoconia from the supporting cells of the maculae is suggested.

Electron Probe X-ray Microanalysis of Human Skeletal Muscle Involved in Rheumatoid Arthritis

SummaryElectron probe X-ray microanalysis in the scanning microscope was used to determine the elemental composition of muscle fibes from patients with rheumatoid arthritis (RA). Quantitative data concerning phosphorus, sulphur, chlorine and potassium were correlated to the fibre type by a routine method based on serial cryosectioning and histochemical staining of adjacent sections. Significantly lowered sulphur values were found in type II A and II B muscle fibres of RA patients as compared to those of healthy controls. Traces of gold were detected in muscles of two patients treated with gold salts. The basis and mechanism for the decreased sulphur content in RA muscles are so far unknown, but may depend on the decreased amount of sulphur-rich protein(s).

Deterioration of the elemental composition of endolymph in genetic inner ear disease

SummaryA normal elemental composition of the endolymph in the scala media of the Shaker-1 and the Shaker-2 mouse does not occur even when there is a normal cell morphology of the stria vascularis. In addition to distinct peaks for potassium and chlorine — but with a decreased relative peak intensity as compared with normals — sodium, phosphorus, sulphur, and calcium were detected. In normal cochlear endolymph only potassium and chlorine were found. At a point of time when the dark cells of the stria vascularis showed severe atrophy, the deterioration of the elemental content of endolymph in the cochlea had further increased showing low concentrations of potassium and chlorine.The dark cell region around vestibular organs remained ultrastructurally rather intact in several months old animals but could reveal an extensive vacuolization of cell cytoplasm and occasionally a reduced number of intercellular extensions towards the basal membrane. The elemental analysis of vestibular endolymph revealed reduced but still high peaks for chlorine and potassium and a distinct peak for sodium thus having an endolymph-like elemental composition. The secretory/reabsorptive epithelia in the vestibular part of the inner ear thus are either not sufficient alone to maintain the specific ionic composition of endolymph or have an inborn type of metabolic disorder in ultrastructurally rather normal cells and therefore are unable to produce a normal composition of endolymph.The fluid filled spaces of the homozygotic Kreisler mouse inner ear did not reveal an elemental composition similar to endolymph or even to a modified type of endolymph.

Development of endolymph during maturation of the mammalian inner ear

SummaryThe development of the elemental composition in the endolymphatic space was investigated during embryologic and early post natal maturation of the CBA/CBA mouse. At birth the elemental distribution was similar in the endo- and perilymphatic spaces. Mature composition of endolymph was reached 6–8 days post partum. The maturation of endolymph corresponded well in time with the morphological maturation of the stria vascularis.