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Ratio confocal imaging of free cytoplasmic calcium gradients in polarising and polarised Fucus zygotes

Published online by Cambridge University Press:  26 September 2008

Frederic Berger  and
Colin Brownlee
Frederic Berger*
Affiliation:
Ecole Normale Suprieure de Lyon, France and Marine Biological Association, Plymouth, UK
Colin Brownlee*
Affiliation:
Ecole Normale Suprieure de Lyon, France and Marine Biological Association, Plymouth, UK
*
Colin Brownlee, Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK. Telephone: (0752) 222772. Fax: (0752) 226865.
Colin Brownlee, Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK. Telephone: (0752) 222772. Fax: (0752) 226865.
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Summary

In the marine brown alga, Fucus, two poles are differentiated before cell division determining the future rhizoid or thallus. We have used a combination of the Ca2+ -sensitive dye Calcium Green and the pH-sensitive dye SNARF monitored at pH-insensitive wavelengths to obtain confocal ratio images of free cytoplasmic calcium distribution at different stages in polarising Fucus zygotes. These dyes have the advantage that they can be used in most confocal microscopes and their longer excitation wavelengths greatly reduce autofluorescence problems. Dyes of varying molecular weights (free acid form, 10 000 mol.wt or 70 000 mol.wt dextran-conjugated) were pressure microinjected into early zygotes which were allowed to polarise in unidirectional light. Dextran-conjugated dyes remained non-compartmentalised and fluorescence could be monitored for up to 3 days following microinjection. Currently we have been able to detect Ca2+ gradients at the tip of the rhizoid, confirming earlier results. Localised Ca2+ elevations have also been observed at the rhizoid pole of the polarising zygote before the onset of rhizoid germination. Limitations of this technique and the significance of these Ca2+ gradients are discussed.

Keywords

Confocal microscopyratio imagingCytoplasmic Ca2+Fucus serratusPolarization
Type
Article
Information
Zygote , Volume 1 , Issue 1 , February 1993 , pp. 9 - 15
Copyright
Copyright © Cambridge University Press 1993

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