Imaging cytosolic free-calcium distribution and oscillations in pollen tubes with confocal microscopy:: a comparison of different dyes and loading methods

A sleep, oscillating tip-focused gradient in cytosolic free calcium ([Ca2+](c)) has been implicated in pollen tube growth. Further understanding of the biological causes and consequences of these processes relies on the precise imaging of [Ca2+](c) during the differ ent growth phases. In this work, the minimum technical requirements for confocal [Ca2+](c) imaging of Agapanthus umbellatus pollen tubes were examined. A range of dyes dye forms, and loading methods were compared. Non-ratio and ratio imaging were critically analysed, in terms of the detection of the [Ca2+](c) gradient and its fluctuations over time. Both ratiometric and nonratiometric methods detected relative changes in [Ca2+](c). However, visualisation of the [Ca2+](c) gradient, with an accurate spatial definition, was only possible with ratiometric methods. The gradient observed in this study ranged from 1.8 mu M (tip) to 180-220 nM (basal level),within the first 4-10 mu m. Apical [Ca2+](c) fluctuations with an amplitude between 415 nM and 1.8 mu M showed a period of 40 to 75 s. All protocols for dye-loading proved to have strengths and weaknesses. Thus, the choice of a dye and its loading procedure should consider the required imaging period, extent of sequestration, effect on cell performance and viability, ease of loading procedure, and aim of the study. The present study constitutes an examination of the [Ca2+](c) gradient in pollen tubes by these criteria.