Cryofixation of Diffusible Elements in Cells and Tissues for Electron Probe Microanalysis

  • Karl Zierold
  • Rudolf Alexander Steinbrecht


The interaction of the electron beam with the specimen not only provides information on its ultrastructure, but also on its elemental composition. In principle, the irradiating electron beam (probe) is focussed on the area of interest in the specimen, while the secondary radiation (e.g. X-rays) generated by the interaction with the specimen is processed through a spectrometer to provide information on the kind and amount of the elements present. Today, electron probe X-ray microanalysis (EPXMA) is the most widely used method in biological microanalysis. Also, electron energy loss spectrometry (EELS) acquires more and more attention. It would be beyond the scope of this book to explain the physical principles and the methodology of these analytical methods here, in particular, since there are numerous, well-written reviews on this subject (e.g. Chandler 1977; Hren et al. 1979; Hall and Gupta 1983; Morgan 1985). Rather, we want to discuss the particular importance of cryotechniques in this field and to point out the still existing problems of specimen preparation. Its crucial steps are not only relevant for EPXMA and EELS, but also for other microanalytical techniques such as proton probe X-ray microanalysis and laser probe mass spectrometry.


Electron Probe Microanalysis Electron Energy Loss Spectrometry Frog Skin Diffusible Element Bulk Specimen 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. Acker H, Pietruschka F, Zierold K (1985) Comparative measurements of postassium and chloride with ion-sensitive microelectrodes and X-ray microanalysis in cultured skeletal muscle fibers. In Vitro Cell Dev Biol 21:45–48.PubMedCrossRefGoogle Scholar
  2. Beck F, Dörge A, Rick R, Thurau K (1985) Osmoregulation of renal papillary cells. Pflügers Arch 405 (Suppl 1):s28–s32.PubMedCrossRefGoogle Scholar
  3. Buja LM, Hagler HK, Parsons D, Chien K, Reynolds RC, Willerson JT (1985) Alterations of ultrastructure and elemental composition in cultured neonatal rat cardiac myocytes after metabolic inhibition with iodoacetic acid. Lab Invest 53:397–412.PubMedGoogle Scholar
  4. *.
    Chandler JA (1977) X-ray microanalysis in the electron microscope. Elsevier/North-Holland Biomedical Press, Amsterdam.Google Scholar
  5. Dörge A, Gehring K, Nagel W, Thurau K (1974) Localization of sodium in frog skin by electron microprobe analysis. Naunyn-Schmiedeberg’s Arch Pharmacol 281:271–280.CrossRefGoogle Scholar
  6. Dörge A, Rick R, Beck F, Thurau K (1985) Cl transport across the basolateral membrane in frog skin epithelium. Pflügers Arch 405 (Suppl 1):s8–s11.PubMedCrossRefGoogle Scholar
  7. Dow JAT, Gupta BL, Hall TA, Harvey WR (1984) X-ray microanalysis of elements in frozen-hydrated sections of an electrogenic K+ transport system: The posterior midgut of tobacco hornworm (Manduca sexta) in vivo and in vitro. J Membr Biol 77:223–241.PubMedCrossRefGoogle Scholar
  8. *.
    Edelmann L (1986) Freeze-dried embedded specimens for biological microanalysis. Scanning Electron Microsc 1986/IV:1337–1356Google Scholar
  9. *.
    Gupta BL (1984) Models of salt and water flow across epithelia: an evaluation by electron probe X-ray microanalysis. In: Pequeux A, Gilles R, Bolis L (eds) Osmoregulation in estuarine and marine animals. Springer, Berlin Heidelberg New York, pp 191–211.Google Scholar
  10. *.
    Gupta BL, Hall TA (1978) Electron microprobe X-ray analysis of calcium. Ann N Y Acad Sci 307:28–51.CrossRefGoogle Scholar
  11. *.
    Gupta BL, Hall TA (1982) Electron probe X-ray microanalysis. In: Baker PF (ed) Techniques in cellular physiology, pt 2, P128. Elsevier/North-Holland Biomedical Press, Amsterdam, pp 1–52.Google Scholar
  12. Gupta BL, Hall TA (1983) Ionic distribution in dopamine-stimulated NaCl fluid-secreting cockroach salivary glands. Am J Physiol 244:R176–R186.PubMedGoogle Scholar
  13. Gupta BL, Hall TA (1984) Role of high concentrations of Ca, Cu, and Zn in the maturation and discharge in situ of sea anemone nematocysts as shown by X-ray microanalysis of cryosections. In: Bolis L, Zadunaisky J, Gilles R (eds) Toxins, drugs, and pollutants in marine animals. Springer, Berlin Heidelberg New York, pp 77–95.CrossRefGoogle Scholar
  14. Gupta BL, Hall TA, Maddrell SHP, Moreton RB (1976) Distribution of ions in a fluid-transporting epithelium determined by electron-probe X-ray microanalysis. Nature (London) 264:284–287.CrossRefGoogle Scholar
  15. Gupta BL, Berridge MJ, Hall TA, Moreton RB (1978) Electron microprobe and ion-selective microelectrode studies of fluid secretion in the salivary glands of Calliphora. J Exp Biol 72:261–284.PubMedGoogle Scholar
  16. Hagler HK, Buja LM (1984) New techniques for the preparation of thin freeze dried cryosections for X-ray microanalysis. In: Revel J-P, Barnard T, Haggis GH (eds) The science of biological specimen preparation. SEM, AMF O’Hare, IL 60666, pp 161–166.Google Scholar
  17. Hall TA (1986a) Properties of frozen sections relevant to quantitative microanalysis. J Microsc (Oxford) 141:319–328.CrossRefGoogle Scholar
  18. *.
    Hall TA (1986b) The history and the current status of biological electron-probe X-ray microanalysis. Micron Microsc Acta 17:91–100.CrossRefGoogle Scholar
  19. Hall TA, Gupta BL (1979) EDS quantitation and application to biology. In: Hren JJ, Goldstein JI, Joy DC (eds) Introduction to analytical electron microscopy. Plenum, New York, pp 169–197.Google Scholar
  20. *.
    Hall TA, Gupta BL (1982) Quantification for the X-ray microanalysis of cryosections. J Microsc (Oxford) 126:333–345.CrossRefGoogle Scholar
  21. *.
    Hall TA, Gupta BL (1983) The localization and assay of chemical elements by microprobe methods. Q Rev Biophys 16:279–339.PubMedCrossRefGoogle Scholar
  22. *.
    Hall TA, Gupta BL (1984) The application of EDXS to the biological sciences. J Microsc (Oxford) 136:193–208.CrossRefGoogle Scholar
  23. Hall TA, Höhling HJ (1969) The application of microprobe analysis to biology. In: Möllenstedt G, Gaukler KH (eds) X-ray optics and microanalysis. Springer, Heidelberg Berlin New York, pp 582–591.Google Scholar
  24. *.
    Harvey DMR (1982) Freeze substitution. J Microsc (Oxford) 127:209–221.CrossRefGoogle Scholar
  25. *.
    Hren JJ, Goldstein JI, Joy DC (1979) Introduction to analytical electron microscopy. Plenum, New York.Google Scholar
  26. Ingram MJ, Ingram FD (1983) Electron microprobe calibration for measurement of intracellular water. Scanning Electron Microsc 1983/III:1249–1254.Google Scholar
  27. Jones RT, Johnson RT, Gupta BL, Hall TA (1979) The quantitative measurement of electrolyte elements in nuclei of maturing erythrocytes of chick embryo using electron-probe X-ray microanalysis. J Cell Sci 35:67–85.PubMedGoogle Scholar
  28. Larsson L, Aperia A, Lechene C (1986) Ionic transport in individual renal epithelial cells from adult and young rats. Acta Physiol Scand 126:321–332.PubMedCrossRefGoogle Scholar
  29. Lubbock R, Gupta BL, Hall TA (1981) Novel role of calcium in exocytosis: Mechanism of nematocyst discharge as shown by X-ray microanalysis. Proc Natl Acad Sci USA 78:3624–3628.PubMedCrossRefGoogle Scholar
  30. *.
    Marshall AT (1980a) Freeze-substitution as a preparation technique for biological X-ray microanalysis. Scanning Electron Microsc 1980/II:395–408Google Scholar
  31. *.
    Marshall AT (1980b) Frozen-hydrated bulk specimens. In: Hayat MA (ed) X-ray microanalysis in biology. Univ Park Press, Baltimore, pp 167–196.Google Scholar
  32. Marshall AT, Hyatt AD, Phillips JG, Condron RJ (1985) Isosmotic secretion in the avian nasal salt gland: X-ray microanalysis of luminal and intracellular ion distributions. J Comp Physiol B 156:213–227.CrossRefGoogle Scholar
  33. Meyer R, Schmitz M, Zierold K (1985) The influence of different cryopreparations on the distribution of ions in bullfrog myocard cells. Scanning Electron Microsc 1985/I:419–431Google Scholar
  34. *.
    Moreton RB (1981) Electron-probe X-ray microanalysis: Techniques and recent applications in biology. Biol Rev 56:409–461.PubMedCrossRefGoogle Scholar
  35. *.
    Morgan AJ (1985) X-ray microanalysis in electron microscopy for biologists. Univ Press, OxfordGoogle Scholar
  36. Rick R, Beck FX, Dörge A, Thurau K (1985) Electron microprobe analysis of chloride secretion in the frog cornea. Current Eye Res 4:377–384.CrossRefGoogle Scholar
  37. Roos N, Barnard T (1985) A comparison of subcellular element concentrations in frozen-dried, plastic-embedded, dry-cut sections and frozen-dried cryosections. Ultramicroscopy 17:335–344.PubMedCrossRefGoogle Scholar
  38. Sasaki S, Nakagaki I, Mori H, Imai Y (1983) Intracellular calcium store and transport of elements in acinar cells of the salivary gland determined by electron probe X-ray microanalysis. Jap J Physiol 33:69–83.CrossRefGoogle Scholar
  39. Schmitz M, Meyer R, Zierold K (1985) X-ray microanalysis in cryosections of natively frozen Paramecium caudatum with regard to ion distribution in ciliates. Scanning Electron Microsc 1985/I:433–445.Google Scholar
  40. Somlyo AP, Bond M, Somlyo AV (1985) Calcium content of mitochondria and endoplasmic reticulum in liver frozen rapidly in vivo. Nature (London) 314:622–625.CrossRefGoogle Scholar
  41. Somlyo AV, Shuman H, Somlyo AP (1977) Elemental distribution in striated muscle and the effects of hypertonicity — Electron probe analysis of cryo sections. J Cell Biol 74:828–857.PubMedCrossRefGoogle Scholar
  42. Somlyo AV, McClellan G, Gonzalez-Serratos H, Somlyo AP (1985) Electron probe X-ray microanalysis of post-tetanic Ca2+ and Mg2+ movements across the sarcoplasmic reticulum in situ. J Biol Chem 260:6801–6807.PubMedGoogle Scholar
  43. Warley A, Stephen J, Hockaday A, Appleton TC (1983) X-ray microanalysis of HeLa S3 cells. II. Analysis of elemental levels during the cell cycle. J Cell Sci 62:339–350.PubMedGoogle Scholar
  44. *.
    Wendt-Gallitelli M-F, Wolburg H (1984) Rapid freezing, cryosectioning, and X-ray microanalysis on cardiac muscle preparations in defined functional states. J Electron Microsc Tech 1:151–174.CrossRefGoogle Scholar
  45. Wróblewski J, Wróblewski R (1986) Why low temperature embedding for X-ray microanalytical investigations? A comparison of recently used preparation methods. J Microsc (Oxford) 142:351–362.CrossRefGoogle Scholar
  46. Yarom R, Hall TA, Stein H, Robin GC, Makin M (1973) Identification and localization of intraarticular colloid gold: ultrastructural and electron probe examinations of human biopsies. Virchows Arch Abt B Cell Pathol 15:11–22.Google Scholar
  47. Zglinicki T von, Bimmler M, Purz H-J (1986) Fast cryofixation technique for X-ray microanalysis. J Microsc (Oxford) 141:79–90.CrossRefGoogle Scholar
  48. *.
    Zierold K (1983) X-ray microanalysis of frozen-hydrated specimens. Scanning Electron Microsc 1983/II:809–826.Google Scholar
  49. *.
    Zierold K (1986a) Preparation of cryosections for biological microanalysis. In: Müller M, Becker RP, Boyde A, Wolosewick JJ (eds) The science of biological specimen preparation 1985. SEM, AMF O’Hare, IL 60666, pp 119–127.Google Scholar
  50. Zierold K (1986b) The determination of wet weight concentrations of elements in freeze-dried cryosections from biological cells. Scanning Electron Microsc 1986/II:713–724.Google Scholar
  51. Zierold K, Schäfer D (1983) Röntgenmikroanalyse diffundibler Substanzen im Ultradünnge-frierschnitt. Acta Histochem Suppl 28:63–72.PubMedGoogle Scholar
  52. Zierold K, Schäfer D, Pietruschka F (1984) The element distribution in ultrathin cryosections of cultivated fibroblast cells. Histochemistry 80:333–337.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1987

Authors and Affiliations

  • Karl Zierold
    • 1
  • Rudolf Alexander Steinbrecht
    • 2
  1. 1.Max-Planck-Institut für SystemphysiologieDortmundGermany
  2. 2.Max-Planck-Institut für VerhaltensphysiologieSeewiesenGermany

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