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Preparation of freeze-dried cryosections for quantitative X-ray microanalysis of electrolytes in biological soft tissues

  • Transport Processes, Metabolism and Endocrinology; Kidney, Gastrointestinal Tract, and Exocrine Glands
  • Instruments and Techniques
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Summary

A procedure is described which allows the evaluation of wet weight concentrations of diffusible substances in biological soft tissue on a cellular level by the use of energy dispersive X-ray microanalysis. Epithelia of frog skin and toad urinary bladder were used to prepare freeze-dried cryosections without the use of chemical fixatives, cryoprotectants, floating solutions or coating materials.

The specimens were shock-frozen inl-propane (−180°C), cryosectioned (−80°C), sandwiched between collodion films and freeze-dried (−80°C). The analysis was performed in a scanning electron microscope at an acceleration voltage of 15 kV, probe current of 0.5 nA, using scanning areas of 1–2 μm2. The spatial resolution power using 1–2 μm thick sections was about 0.7 μm. In a superficial layer of about 30 μm the analysis was found not to be influenced by tissue damage due to ice crystal formation. The mass loss during electron bombardement was restricted to constituents of the organic matrix (30%). No changes of characteristic radiation were observed for Na, K and Cl. Albumin standards, containing electrolyte concentrations in the range of biological interest, revealed linear calibration curves. To obtain reliable wet weight concentrations, the characteristic X-rays of the tissue were compared to those of an internal standard which was applied to the specimen prior to freezing and analysed simultaneously.

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Authors and Affiliations

  1. Institut für Physiologie, Universität München, Pettenkoferstrasse 12, D-8000, München 2, Federal Republic of Germany

    A. Dörge, R. Rick, K. Gehring & K. Thurau

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  1. A. Dörge
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  4. K. Thurau
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Dörge, A., Rick, R., Gehring, K. et al. Preparation of freeze-dried cryosections for quantitative X-ray microanalysis of electrolytes in biological soft tissues. Pflugers Arch. 373, 85–97 (1978). https://doi.org/10.1007/BF00581153

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