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

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, l0000 mol.wt or 70000 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.