Quantitative X-ray microanalysis of semi-thick cryosections

Abstract

Methodological aspects of quantitative X-ray microanalysis of semi-thick cryosections (2--6 micrometers) 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. Factor 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).

Quantitative correlative proton and electron microprobe analysis of biological specimens

“…This method, which has also been used in EMP analysis (Zs. Nagy et al 1976) may cause minor errors that can be corrected for by taking the difference in absorption in two spectral regions into account (Wroblewski et al 1983). In the particular case studied in this paper, this effect is, however, negligible, since both background and characteristic intensities were determined at relatively high X-ray energies.…”

Effect of luminal osmolarity on ion content of connective tissue in rat trachea after epithelial damage

“…Determination of elemental content in the subepithelial connective tissue X-ray microanalysis was used to quantify the ion content in the subepithelial CT. Tissue pieces were cut into semithin (3-4 mm) cryosections in a conventional cryostat at -30uC [12], mounted on a carbon specimen holder over a layer of thin Formvar film (Merck, Darmstadt, Germany) and freeze-dried for 24 h. To avoid charging in the electron microscope, the sections were coated with a conductive carbon layer after the specimen was successively warmed to room temperature. The sections were analysed in the scanningtransmission electron microscope mode at 100 kV (Hitachi 7100, Hitachi, Tokyo, Japan) with an Oxford Instruments ISIS energy-dispersive spectrometer system (Oxford Instruments, Oxford, UK).…”

Low temperature techniques for X‐ray microanalysis in pathology: Alternatives to cryoultramicrotomy

“…For analysis at high resolution, thin sections prepared by cryoultramicrotomy or by sectioning of freeze-dried embedded or freeze-substituted embedded material have to be used. For analysis at lower resolution, thick or semithick cryosections cut on a conventional cryostat at temperatures between -20 and -35°C can be used (Wroblewski et al, 1983b;Wrdblewski, 1982;Wrdblewski et al, 1978). Freeze-dried embedded bulk specimens (Ingram and Ingram, 1980) or frozen-hydrated bulk specimens (Marshall, 1980a;Zs.…”