Hostname: page-component-78c5997874-j824f Total loading time: 0 Render date: 2024-11-10T08:22:59.668Z Has data issue: false hasContentIssue false
  • English
  • Français

The Distribution of Light Elements in Biological Cells Measured by Electron Probe X-Ray Microanalysis of Cryosections

Published online by Cambridge University Press:  08 March 2005

Karl Zierold ,
Jean Michel ,
Christine Terryn  and
Gérard Balossier
Karl Zierold
Affiliation:
Max-Planck-Institute of Molecular Physiology, 44227 Dortmund, Germany
Jean Michel
Affiliation:
INSERM ERM 203, University of Reims, 51685 Reims, France
Christine Terryn
Affiliation:
INSERM ERM 203, University of Reims, 51685 Reims, France
Gérard Balossier
Affiliation:
INSERM ERM 203, University of Reims, 51685 Reims, France
Get access

Abstract

The intracellular distribution of the elements carbon, nitrogen, and oxygen was measured in cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of 100-nm-thick freeze-dried cryosections. Electron irradiation with a dose up to 106 e/nm2 caused no or merely negligible mass loss in mitochondria and in cytoplasm. Cell nuclei lost carbon, nitrogen, and—to a clearly higher extent—oxygen with increasing electron irradiation. Therefore, electron doses less than 3 × 105 e/nm2 were used to measure the subcellular compartmentation of carbon, nitrogen, and oxygen in cytoplasm, mitochondria, and nuclei of the cells. The subcellular distribution of carbon, nitrogen, and oxygen reflects the intracellular compartmentation of various biomolecules. Cells exposed to inorganic mercury before cryofixation showed an increase of oxygen in nuclei and cytoplasm. Concomitantly the phosphorus/nitrogen ratio decreased in mitochondria. The data suggest mercury-induced production of ribonucleic acid (RNA) and decrease of adenosine triphosphate (ATP). Although biomolecules cannot be identified by X-ray microanalysis, measurements of the whole element spectrum including the light elements carbon, nitrogen, and oxygen can be useful to study specific biomolecular activity in cellular compartments depending on the functional state of the cell.

Keywords

cryosectionhepatocytelight elementsmercury cytotoxicityradiation damageX-ray microanalysis
Type
BIOLOGICAL APPLICATIONS
Information
Microscopy and Microanalysis , Volume 11 , Issue 2 , April 2005 , pp. 138 - 145
Copyright
© 2005 Microscopy Society of America

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Cantino, M.E., Wilkinson, L.E., Goddard, M.K., & Johnson, D.E. (1986). Beam induced mass loss in high resolution biological microanalysis. J Microsc 144, 317–327.Google Scholar
Dubochet, J., Chang, J.-J., Freeman, R., Lepault, J., & McDowall, A.W. (1982). Frozen aqueous suspensions. Ultramicroscopy 10, 55–62.Google Scholar
Egerton, R.F. (1982). Organic mass loss at 100 K and 300 K. J Microsc 126, 95–100.Google Scholar
Egerton, R.F. & Rauf, I. (1999). Dose-rate dependence of electron-induced mass loss from organic specimens. Ultramicroscopy 80, 247–254.Google Scholar
Frausto da Silva, J.J.R. & Williams, R.J.P. (1991). The Biological Chemistry of the Elements. The Inorganic Chemistry of Life. Oxford, UK: Clarendon Press.
Goering, P.L., Fisher, B.R., Noren, B.T., Papaconstantinou, A., Rojko, J.L., & Marler, R.J. (2000). Mercury induces regional and cell-specific stress protein expression in rat kidney. Toxicol Sci 53, 447–457.Google Scholar
Hall, T.A. & Gupta, B.L. (1983). The localization and assay of chemical elements by microprobe methods. Quart Rev Biophys 16, 279–339.Google Scholar
Hamer, D.H. (1986). Metallothionein. Ann Rev Biochem 55, 913–951.Google Scholar
Hüttermann, J. (1982). Solid-state radiation chemistry of DNA and its constituents. Ultramicroscopy 10, 25–40.Google Scholar
Isaacson, M. & Johnson, D. (1975). The microanalysis of light elements using transmitted energy loss electrons. Ultramicroscopy 1, 33–52.Google Scholar
Knapek, E. (1982). Properties of organic specimens and their supports at 4K under irradiation and electron microscope. Ultramicroscopy 10, 71–86.Google Scholar
Knapek, E., Lefranc, G., Heide, H.G., & Dietrich, I. (1982). Electron microscopical results on cryoprotection of organic materials obtained with cold stages. Ultramicroscopy 10, 105–110.Google Scholar
Kramer, K., Liu, J., Choudhuri, S., & Klaassen, C.D. (1996). Induction of metallothionein mRNA and protein in murine astrocyte cultures. Toxicol Appl Pharmacol 136, 94–100.Google Scholar
Lamvik, M.K., Kopf, D.A., & Davilla, S.D. (1987). Mass loss rate in collodion is greatly reduced at liquid helium temperature. J Microsc 148, 211–217.Google Scholar
Laquerrière, P., Banchet, V., Michel, J., Zierold, K., Balossier, G., & Bonhomme, P. (2001). X-ray microanalysis of organic thin sections in TEM using an UTW Si(Li) detector: Comparison of quantification methods. Microsc Res Techn 52, 231–238.Google Scholar
Laquerrière, P., Michel, J., Balossier, G., & Chénais, B. (2002). Light elements quantification in stimulated cells cryosections studied by electron probe microanalysis. Micron 33, 597–603.Google Scholar
Leapman, R.D. (2003). Detecting single atoms of calcium and iron in biological structures by electron energy-loss spectrum-imaging. J Microsc 210, 5–15.Google Scholar
Leapman, R.D., Hunt, J.A., Buchanan, R.A., & Andrews, S.B. (1993). Measurement of low calcium concentrations in cryosectioned cells by parallel EELS-mapping. Ultramicroscopy 49, 225–234.Google Scholar
Leapman, R.D. & Ornberg, R.L. (1988). Quantitative electron energy loss spectroscopy in biology. Ultramicroscopy 24, 251–268.Google Scholar
LeFurgey, A., Bond, M., & Ingram, P. (1988). Frontiers in electron probe microanalysis: Application to cell physiology. Ultramicroscopy 9, 19–43.Google Scholar
Michel, J., Sauerwein, W., Wittig, A., Balossier, G., & Zierold, K. (2003). Subcellular localization of boron in cultured melanoma cells by electron energy loss spectroscopy of freeze-dried cryosections. J Microsc 210, 25–34.Google Scholar
Nieminen, A.-L., Gores, G.J., Dawson, T.L., Herman, B., & Lemasters, J.J. (1990). Toxic injury from mercuric chloride in rat hepatocytes. J Biol Chem 265, 2399–2408.Google Scholar
Palmeira, C.M. & Madeira, V.M.C. (1997). Mercuric chloride toxicity in rat liver mitochondria and isolated hepatocytes. Environ Toxicol Pharmacol 3, 229–235.Google Scholar
Razskazovskiy, Y., Debije, M.G., & Bernhard, W.A. (2000). Direct radiation damage to crystalline DNA: What is the source for unaltered base release? Radiat Res 153, 436–441.Google Scholar
Reimer, L. (1984). Methods of detection of radiation damage in electron microscopy. Ultramicroscopy 14, 291–304.Google Scholar
Somlyo, A.V., Shuman, H., & Somlyo, A.P. (1989). Electron probe X-ray microanalysis of Ca2+, Mg2+ and other ions in rapidly frozen cells. Methods Enzymol 172, 203–229.Google Scholar
Sun, S.Q., Shi, S.L., Hunt, J.A., & Leapman, R.D. (1995). Quantitative water mapping of cryosectioned cells by electron energy loss spectrometry. J Microsc 177, 18–30.Google Scholar
Sutton, D.J., Tchounwou, P.B., Ninashvili, N., & Shen, E. (2002). Mercury induces cytotoxicity and transcriptionally activates stress genes in human liver carcinoma (HepG2) cells. Int J Mol Sci 3, 965–984.Google Scholar
Symons, M.C.R. (1982). Chemical aspects of electron-beam interactions in the solid state. Ultramicroscopy 10, 15–24.Google Scholar
Talmon, Y. (1987). Electron beam radiation damage to organic and biological cryospecimens. In Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. & Zierold, K. (Eds.), pp. 64–84. Berlin, Heidelberg: Springer-Verlag.
Terryn, C., Michel, J., Kilian, L., Bonhomme, P., & Balossier, G. (2000). Comparison of intracellular water content measurements by dark-field imaging and EELS in medium voltage TEM. Eur Phys J Appl Phys 11, 215–226.Google Scholar
Zierold, K. (1988). X-ray microanalysis of freeze-dried and frozen-hydrated cryosections. J Electr Microsc Techn 9, 65–82.Google Scholar
Zierold, K. (1997). Effects of cadmium on electrolyte ions in cultured rat hepatocytes studied by X-ray microanalysis of cryosections. Toxicol Appl Pharmacol 144, 70–76.Google Scholar
Zierold, K. (2000). Heavy metal cytotoxicity studied by electron probe X-ray microanalysis of cultured rat hepatocytes. Toxicol in Vitro 14, 557–563.Google Scholar
Zierold, K. (2002). Limitations and prospects of biological electron probe X-ray microanalysis. J Trace Microprobe Techn 20, 181–196.Google Scholar