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Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale : preparative and analytical aspects as exemplified with Paramecium Cells

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1999

Autor:innen

Hardt, Martin

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http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-43503
DOI (zitierfähiger Link)
https://doi.org/10.1006/jsbi.1999.4188
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Attribution-NonCommercial-NoDerivs 2.0 Generic

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Open Access Green

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Biologie: Publikationen
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Journal of Structural Biology. 1999, 128(2), pp. 187-199. ISSN 1047-8477. Available under: doi: 10.1006/jsbi.1999.4188

Zusammenfassung

We analyzed preparative and analytical aspects of the dynamic localization of Ca2+ during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding. The quenched-flow used allowed analyses, during stimulation, in the subsecond time range. Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable. We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (~100-nm thickness) or cilia (~250-nm diameter). We used widely different standards to calibrate the ratio of CaK net counts in relation to actual [Ca]. Calibration curves showed a linear relationship and a detection limit of [Ca] = 2 mM, while [Ca] in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr2+ can rapidly be substituted for Ca2+ in the medium before and during stimulation, thus allowing one to determine Me2+ fluxes. With our ''model'' cell, Paramecium, we showed that, upon stimulation (causing rapid Ca2+ mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.

Zusammenfassung in einer weiteren Sprache

Fachgebiet (DDC)
570 Biowissenschaften, Biologie

Schlagwörter

calcium, cilia, EDX, electron microscopy, exocytos, Paramecium, quenched-flow, secretion

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ISO 690HARDT, Martin, Helmut PLATTNER, 1999. Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale : preparative and analytical aspects as exemplified with Paramecium Cells. In: Journal of Structural Biology. 1999, 128(2), pp. 187-199. ISSN 1047-8477. Available under: doi: 10.1006/jsbi.1999.4188
BibTex
@article{Hardt1999Quant-8546,
  year={1999},
  doi={10.1006/jsbi.1999.4188},
  title={Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale : preparative and analytical aspects as exemplified with Paramecium Cells},
  number={2},
  volume={128},
  issn={1047-8477},
  journal={Journal of Structural Biology},
  pages={187--199},
  author={Hardt, Martin and Plattner, Helmut}
}
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    <dcterms:abstract xml:lang="eng">We analyzed preparative and analytical aspects of the dynamic localization of Ca2+ during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding. The quenched-flow used allowed analyses, during stimulation, in the subsecond time range. Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable. We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (~100-nm thickness) or cilia (~250-nm diameter). We used widely different standards to calibrate the ratio of CaK net counts in relation to actual [Ca]. Calibration curves showed a linear relationship and a detection limit of [Ca] = 2 mM, while [Ca] in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr2+ can rapidly be substituted for Ca2+ in the medium before and during stimulation, thus allowing one to determine Me2+ fluxes. With our ''model'' cell, Paramecium, we showed that, upon stimulation (causing rapid Ca2+ mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.</dcterms:abstract>
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