An optimized pipeline for live imaging whole Arabidopsis leaves at cellular resolution

Abstract

Live imaging is the gold standard for determining how cellular development gives rise to organs. However, tracking all individual cells across whole organs over large developmental time windows is extremely challenging. In this work, we provide a comparably simple method for confocal live imaging of Arabidopsis thaliana first leaves across early development. Our imaging method works for both wild-type leaves and the complex curved leaves of the jaw-1D mutant. We find that dissecting the cotyledons, affixing a coverslip above the samples and mounting samples with perfluorodecalin yields optimal imaging series for robust cellular and organ level analysis. We provide details of our complementary image processing steps in MorphGraphX software for segmenting cells, tracking the cell lineages, and measuring a suite of cellular growth properties. We also provide MorphoGraphX image processing scripts that we developed to automate analysis of segmented images and data presentation. Our imaging techniques and processing steps combine into a robust imaging pipeline. With this pipeline we are able to examine important nuances in the cellular growth and differentiation of jaw-D versus WT leaves that have not been demonstrated before. Our pipeline is a practical starting place for researchers new to live imaging plant leaves, but also to anyone interested in improving the throughput and reliability of their live imaging process.