Conspicuous intracellular gradients manifest and/or drive intracellular polarity in pollen tubes. However, quantifying these gradients raises multiple technical challenges. Here we present a sensible computational protocol to analyze gradients in growing pollen tubes and to filter nonrepresentative time points. As an example, we use imaging data from pollen tubes expressing a genetically encoded ratiometric Ca2+ probe, Yellow CaMeleon 3.6, from which a kymograph is extracted. The tip of the pollen tube is detected with CHUKNORRIS, our previously published methodology, allowing the reconstruction of the intracellular gradient through time. Statistically confounding time points, such as growth arrest where gradients are highly oscillatory, are filtered out and a mean spatial profile is estimated with a local polynomial regression method. Finally, we estimate the gradient slope by the linear portion of the decay in mean fluorescence, offering a quantitative method to detect phenotypes of gradient steepness, location, intensity, and variability. The data manipulation protocol proposed can be achieved in a simple and efficient manner using the statistical programming language R, opening paths to perform high-throughput spatiotemporal phenotyping of intracellular gradients in apically growing cells.
Keywords: Apical growth; Ca2+; Computational analysis; High-throughput phenotyping; Intracellular gradient; Ion gradients; Ratiometric probes; Spatiotemporal analysis; Tip detection.